Multiple mutation variants of serine protease

ABSTRACT

The present invention provides novel Micrococcineae spp serine proteases having multiple substitutions. In particular, the present invention provides serine proteases having multiple substitutions, DNA encoding these proteases, vectors comprising the DNA encoding the proteases, host cells transformed with the vector DNA, and enzymes produced by the host cells. The present invention also provides cleaning compositions (e.g., detergent compositions), animal feed compositions, and textile and leather processing compositions comprising these serine protease variants. In particularly preferred embodiments, the present invention provides mutant (i.e., variant) proteases derived from the wild-type proteases described herein. These variant proteases also find use in numerous applications.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of U.S. patent application Ser.No. 13/162,104, filed Jun. 16, 2011 (now U.S. Pat. No. 8,455,234), whichis a Divisional of U.S. patent application Ser. No. 11/809,104, filedMay 31, 2007 (now U.S. Pat. No. 7,985,569), which is a Continuation ofU.S. patent application Ser. No. 11/583,334, filed Oct. 19, 2006 (nowAbandoned), which is a Continuation-in-Part of U.S. patent applicationSer. No. 10/576,331, filed Jul. 18, 2007, (now U.S. Pat. No. 8,535,927),which is a 371 National Phase application of PCT/US2004/039066, filedNov. 19, 2004, which claims priority to U.S. Provisional PatentApplication Ser. No. 60/523,609, filed Nov. 19, 2003, the disclosures ofwhich applications are incorporated herein in their entirety.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 C.F.R.§1.52(e), is incorporated herein by reference in it s entirety. Thesequence listing text file submitted via EFS contains the file“931-C1-D1-SEQLIST.txt” created on Aug. 14, 2013, which is 23,601 bytesin size.

FIELD OF THE INVENTION

The present invention provides novel Micrococcineae spp serine proteaseshaving multiple substitutions. In particular, the present inventionprovides serine proteases having multiple substitutions, DNA encodingthese proteases, vectors comprising the DNA encoding the proteases, hostcells transformed with the vector DNA, and enzymes produced by the hostcells. The present invention also provides cleaning compositions (e.g.,detergent compositions), animal feed compositions, and textile andleather processing compositions comprising these serine proteasevariants. In particularly preferred embodiments, the present inventionprovides mutant (i.e., variant) proteases derived from the wild-typeproteases described herein. These variant proteases also find use innumerous applications.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of a diverse class of enzymes having awide range of specificities and biological functions (See e.g., Stroud,Sci. Amer., 131:74-88 [1974]). Despite their functional diversity, thecatalytic machinery of serine proteases is represented by at least twogenetically distinct families of enzymes: 1) the subtilisins; and 2) themammalian chymotrypsin-related and homologous bacterial serine proteases(e.g., trypsins). These two families of serine proteases show remarkablysimilar mechanisms of catalysis (See e.g., Kraut, Ann. Rev. Biochem.,46:331-358 [1977]). Furthermore, although the primary structure isunrelated, the tertiary structure of these two enzyme families bringstogether a conserved catalytic triad of amino acids consisting ofserine, histidine and aspartate.

In contrast, the subtilisins and chymotrypsin-related serine proteasesboth have a catalytic triad comprising aspartate, histidine and serine.In the subtilisin-related proteases the relative order of these aminoacids, reading from the amino to carboxy terminus, isaspartate-histidine-serine. However, in the chymotrypsin-relatedproteases, the relative order is histidine-aspartate-serine. Muchresearch has been conducted on the subtilisins, due largely to theirusefulness in cleaning and feed applications. Additional work has beenfocused on the adverse environmental conditions (e.g., exposure tooxidative agents, chelating agents, extremes of temperature and/or pH)which can adversely impact the functionality of these enzymes in variousapplications. Nonetheless, there remains a need in the art for enzymesystems that are able to resist these adverse conditions and retain orhave improved activity over those currently known in the art.

SUMMARY OF THE INVENTION

The present invention provides novel Micrococcineae spp serine proteaseshaving multiple substitutions. In particular, the present inventionprovides serine proteases having multiple substitutions, DNA encodingthese proteases, vectors comprising the DNA encoding the proteases, hostcells transformed with the vector DNA, and enzymes produced by the hostcells. The present invention also provides cleaning compositions (e.g.,detergent compositions), animal feed compositions, and textile andleather processing compositions comprising these serine proteasevariants. In particularly preferred embodiments, the present inventionprovides mutant (i.e., variant) proteases derived from the wild-typeproteases described herein. These variant proteases also find use innumerous applications.

The present invention provides isolated serine protease variants havingan amino acid sequence comprising at least two amino acid substitutions,wherein the substitutions are made at positions equivalent to thepositions in a Cellulomonas 69B4 protease comprising the amino acidsequence set forth in SEQ ID NO:8. The present invention furtherprovides compositions comprising the isolated serine protease variant(s)having at least two amino acid substitutions, wherein the substitutionsare made at positions equivalent to positions in a Cellulomonas 69B4protease comprising the amino acid sequence set forth in SEQ ID NO:8. Insome preferred embodiments, the compositions comprise at least onevariant serine protease, wherein the variant serine protease hasimmunological cross-reactivity with the serine protease set forth in SEQID NO:8. In some additional preferred embodiments, the sequence of aserine protease variant comprises substitutions at least two amino acidpositions selected from positions 1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 18, 19, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 54, 55, 56,57, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 99,100, 101, 103, 104, 105, 107, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 121, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133,134, 135, 136, 137, 140, 141, 142, 143, 144, 145, 146, 147, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 170, 171, 172, 175, 176, 177, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, and 189, wherein the substitutions are made atpositions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8.

In some preferred embodiments, the serine protease variant comprises atleast two substitutions selected from G12D, R14I, R14L, R14M, R14S,R16I, R16L, R16Q, N24E, N24H, N24M, N24T, N24W, R35E, R35F, R35H, T36S,G49A, G54D, G54L, R61V, A64K, G65Q, Q71F, Y75G, S76A, S76L, S76N, S76T,S76V, R79K, R79T, Q81K, Q81P, T86K, A93G, A93H, A93S, S99A, T109M,N112E, T116E, R123F, R123L, R123Q, R123S, R127A, R127K, R127Q, R159E,R159F, R159G, R159K, R159L, R159Q, R179N, R179Q, I181K, I181Q, I181T,D184N, D184T, and S187Q, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8. In some yet additionalpreferred embodiments, the serine protease variant comprises multiplesubstitutions selected from R16Q/R35F/R159Q, R16Q/R123L,R14L/R127Q/R159Q, R14L/R179Q, R123L/R127Q/R179Q, R16Q/R79T/R127Q,R16Q/R79T, R35E/R123L/R127Q/R179Q, and G12D/R35E/G63R/R79K/T109M,wherein the substitutions are made at positions equivalent to thepositions in a Cellulomonas 69B4 protease comprising the amino acidsequence set forth in SEQ ID NO:8. In some still further embodiments,the serine protease variant comprises the following substitutions R123L,R127Q, and R179Q, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8.

In some further preferred embodiments, the serine protease variantcomprises at least two substitutions selected from D2G, D2Q, V3I, V3L,N7A, N7L, N7S, I11A, 111Q, R14E, N24A, N24E, N24H, N24L, N24M, N24Q,N24T, N24V, T36D, T36F, T36G, T36H, T36I, T36L, T36N, T36P, T36R, T36S,T36V, T36W, T36Y, A38D, A38F, A38H, A38L, A38N, A38R, A38S, G49F, S51A,G54A, G54D, G54H, G54K, G54L, G54M, G54R, N55F, A64H, A64N, A64R, A64W,A64Y, G65L, G65P, G65Q, G65R, G65S, G65T, G65Y, G65V, V66A, V66D, V66E,V66H, V66I, V66L, N67A, N67G, N67L, N67K, L69H, L69S, L69V, A70D, A70H,A705, Q71A, Q71G, Q71H, Q71I, Q71K, Q71M, Q71N, N73S, N73T, N74G, Y75F,Y75G, Y75I, S76L, S76Y, S76V, S76W, G77S, G77T, G78A, G78D, G78H, G78N,G78S, G78T, R79P, V80H, V80L, Q81H, Q81K, Q81V, H85Q, H85T, V90I, V90P,V90S, S92G, W103I, W103M, H104K, T109A, T109H, T109I, S114G, T116F,P118A, P118F, P118H, P118R, E119K, E119R, N145E, N145I, N145Q, V150L,R159F, N170Y, G177M, R179A, R179D, R179E, R179I, R179K, R179L, R179M,R179N, R179T, R179Y, R179V, I181H, T183I, G186E, G186I, G186V, S187P,S187T, and S188M, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8.

In some additional embodiments, the serine protease variant comprises atleast two substitutions selected from F1A, F1T, D2A, D2H, D2N, V3T, N7H,N7I, A8G, A8K, T10G, T10K, I11S, I11T, G12W, G13M, S15F, N24F, N24S,A30S, R35F, T36C, A38G, A38I, A38K, A38V, A38Y, T40S, T40V, A41N, N42H,F47I, F47M, G49A, G49K, G49L, S51F, S51Q, G54I, G54Q, N55K, N55Q, R61M,T62I, G63Q, G63V, G63W, A64F, A64I, A64K, A64L, A64M, A64Q, A64S, A64T,A64V, G65A, G65H, V66M, V66N, N67D, N67F, N67H, N67Q, N67R, N67S, N67T,N67V, N67Y, L68W, L69W, A70G, Q71D, Q71F, Q71L, Q71R, V72I, N73H, S76E,S76I, S76K, S76A, S76N, S76Q, S76R, S76T, G77N, G77Y, G78I, S78R, G78V,R79G, V80F, Q81D, Q81I, A83N, H85R, H85K, H85L, T86A, P89N, V90A, V90L,V90T, T107H, T107M, T107S, T107V, T109G, T109L, T109P, T109R, A110S,A110T, N112I, P118E, P118I, P118K, P118Q, R123E, R123I, I126L, R127F,128L, T129S, E133Q, L142V, A143N, A143S, N145G, N145L, N145T, V150M,T151L, R159E, T163L, Q167N, N170A, N170D, N170L, 171S, G177S, I181G,I181N, T182V, T183K, T183M, D184F, D184H, D184Q, D184R, S185I, S185V,S187E, and S187L, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8.

In yet additional embodiments, the serine protease variant comprises atleast two substitutions selected from D2P, A8G, T10C, T10L, I11E, I11Q,I11T, I11W, G12D, G12I, G12N, G12Q, G12S, G12V, R14A, R14C, R14E, R14D,R14G, R141, R14N, R14Q, R14S, R14T, 515C, 515E, S15H, S15R, S15Y, R16C,R16D, R16E, R16T, R16V, A22C, A22S, N24E, R35A, R35C, R35D, R35E, R35H,R35M, R35N, R35P, R35Q, R35S, R35T, R35V, T36C, A38C, A38D, A41C, A41D,T44E, T46C, T46E, T46F, T46V, T46Y, F47R, A48E, G49A, G49C, G49E, G49H,G49L, G49N, G49Q, G49V, G54C, N55G, D56L, Y57G, F59W, R61E, R61M, R61T,R61V, G91Q, S99A, T100A, T100R, T107R, T109E, N112P, S113C, S114C,P118K, P118R, E119G, E119R, E119T, E119V, E119Y, T121E, T121F, T121L,R123C, R123D, R123E, R123F, R123H, R123N, R123Q, R123S, R123T, R123V,R123W, R123Y, G124D, L125Q, R127D, R127E, R127K, R127Q, R127S, P134R,T151C, T151L, S155C, S155I, S155W, S155Y, R159D, R159E, R159Q, R159S,R159T, R159V, T163D, F165E, F165W, Q167E, N170C, N170D, G177D, R179D,R179E, and M180L, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8. In still furtheradditional embodiments, the serine protease variant comprises at leasttwo substitutions selected from F1N, F1P, D2I, D2M, D2T, D2V, A8R, A8T,T10D, T10E, T10F, T10M, T10Q, T10Y, G12H, G12P, G12Y, G13D, G13E, R14H,R14L, R14M, S15F, S15G, S15N, R16I, R16Q, N24G, N24T, 128V, R35K, T36V,A41S, A41T, N42D, T44C, F47V, G49F, G49K, G49S, S51A, S51C, S51L, S15M,G54E, N55A, D56F, R61K, R61Q, A64C, G65D, V66N, A70G, A70M, A70P, R79T,R79V, Q81A, Q81G, Q81P, A83E, A83D, A83H, T86E, A87C, A87E, A88F, S92T,S99G, S99H, S99K, S99Q, T100K, T100Q, W103L, T109K, N112D, N112E, S113A,S113D, S114E, T115C, T116G, T116N, P118A, P118C, P118G, P118W, E119A,E119L, E119N, E119Q, E119S, T121A, T121D, R123A, R123G, R123I, R123K,R123M, A132S, L125M, R127A, R127C, T128A, S140P, L141M, T151V, S155E,S155F, S155T, S155V, N157D, R159A, R159C, R159K, R159M, R159N, T160D,T163C, F165H, N170L, I172A, Q174C, Q174S, Q174T, A175T, G177E, R179C,R179F, R179I, R179L, R179M, R179N, R179S, R179T, R179V, R179W, R179Y,S187E, and S188E, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8.

In some further embodiments, the present invention provides a serineprotease variant, wherein the amino acid sequence of the proteasecomprises at least two substitutions selected from V3R, I4D, I4G, I4P,Y9E, Y9P, T10F, T10W, T10Y, G12D, S18E, A22C, A22S, A22T, N24T, G26E,G26I, G26K, G26Q, G26V, G26W, F27V, F27W, I28P, I28T, T29E, A30M, A30N,A30P, A30Y, G31H, G31M, G31N, G31V, G31Y, C33E, C33L, C33M, C33N, A38D,A38G, T39R, T40D, T40H, T40N, T40P, T40Q, R43D, P43G, P43H, P43K, P43L,P43N, G45A, G45V, T46V, T46Y, T46W, A48P, Y57M, Y57N, F59K, T62G, T62R,A70G, A70P, N73P, R79T, Q81A, Q81D, Q81F, Q81G, Q81H, Q81P, Q81S, A83H,G84C, G84P, P89W, G91L, A93S, R96C, R96E, R96F, S99A, T100A, C105E,C105G, C105K, C105M, C105N, C105P, C105S, C105W, T121E, R123F, R123N,R123W, R123Y, L125A, T128A, T128C, T128G, T128S, T128V, 128W, T129W,S137R, S140P, Q146P, A147E, S155F, S155K, S155P, S155R, S155W, S155Y,G156I, G156L, G156P, C158G, C158H, C158M, R159K, I160I, G161I, G161L,G161V, T164G, T164L, F166S, Q167L, P168Y, Y176P, G186S, and S188A,wherein the substitutions are made at positions equivalent to thepositions in a Cellulomonas 69B4 protease comprising the amino acidsequence set forth in SEQ ID NO:8. In some embodiments, the amino acidsequence of the serine protease variant comprises at least twosubstitutions selected from A8G, T10C, T10L, G12A, G12H, S15C, S15N,S15Q, S15R, 515T, N24E, N24S, G25S, F27I, G31A, H32A, C33D, T36V, T39V,A41S, T46F, G49A, S51V, F59W, Q71A, Q71Y, N74F, R79V, Q81C, Q81E, A83E,A83F, A83M, A83R, G84M, G84V, T86I, T86M, T86S, A87E, A87S, P89A, V90A,V90M, S92T, A93D, S99G, T100Q, T101S, W103N, C105A, C105L, C105T, C105Y,T107A, T107F, T107L, T107Q, T107S, T110D, A110G, L111K, V115I, V115L,T116Q, Y117K, Y117Q, Y117R, Y117V, P118T, E119L, T121A, T121D, R123I,R123K, R123L, R123Q, R123T, L125M, R127F, R127K, R127Q, T129Y, V130T,A132C, P134W, L141C, A143H, G144A, V150N, T151C, G153K, G153V, G154L,G154R, S155T, R159Q, R159T, R159V, T160E, T160Q, G161K, G162P, T163I,F166A, F166C, P168I, N170D, N170E, G177N, R179K, M180L, T182L, T183A,T183I, T183P, S185R, G186P, S188C, S188E, S188G, S188M, S188T, S188V,and P189S, wherein the substitutions are made at positions equivalent tothe positions in a Cellulomonas 69B4 protease comprising the amino acidsequence set forth in SEQ ID NO:8.

The present invention further provides a serine protease variant,wherein the amino acid sequence of the protease comprises at least twosubstitutions selected from FIT, T10N, R14D, R14G, R14I, R14L, R14N,R14Q, R14T, N24A, N24E, N24H, N24L, N24Q, N24T, N24V, R35A, R35E, R35F,R35L, R35Q, R35T, T36G, T36I, T36N, T36S, A38D, A38F, A38H, A38N, A38R,G49A, G49S, S51D, G54D, G54E, N55E, N55F, A64I, G65D, G65P, G65Q, G65S,G65T, G65V, N67D, L69S, N73T, N74G, Y75F, Y75G, S76D, S76E, S76I, S76L,S76N, S76T, S76V, S76Y, G77T, G78A, G78D, R79A, R79D, R79E, R79G, R79L,R79M, R79P, R79S, R79T, R79V, Q81E, A83E, H85Q, H85T, T86D, T86E, V90I,V90P, V905, V90T, S99N, S99V, T107E, T107H, T107S, T107V, T109E, N112D,N112E, N112L, N112Q, N112V, T116E, T116Q, T121E, R123A, R123D, R123E,R123F, R123H, R123I, R123L, R123N, R123Q, R127A, R127Q, T129S, L142V,N145E, R159D, R159E, R159F, R159N, R159Q, N170D, N170Y, I172T, R179A,R179D, R179E, R179I, R179K, R179M, R179N, R179T, R179V, R179Y, I181L,and G186N, wherein the substitutions are made at positions equivalent tothe positions in a Cellulomonas 69B4 protease comprising the amino acidsequence set forth in SEQ ID NO:8. In some alternative embodiments, theamino acid sequence of the protease comprises at least two substitutionsselected from F1D, V3L, N7L, A8E, A8G, T10D, T10E, G12D, G13S, R14A,R14K, R14S, R14M, 515W, I19V, N24M, R35H, R35M, R35S, R35W, R35Y, T36D,T36H, A38L, A38S, A38T, A38Y, T40V, A41D, A41N, A48E, G49F, G49H, S51H,S51Q, S51T, S51V, R61E, R61H, R61M, R61S, R61T, G63D, A64F, A64H, A64L,A64M, A64N, A64P, A64Q, A64S, A64T, A64V, A64W, A64Y, G65L, G65Y, N67E,N67G, N67H, N67S, N67T, A70D, A70G, A70H, Q71D, Q71G, Q71H, Q71S, V72I,S76Q, S76W, G77S, Q81D, Q81H, Q81V, H85L, H85M, V90N, S92A, S92G, A93D,A93E, A93S, S99D, S99T, T101S, W103M, T107A, T107I, T107M, T107N, T109A,T109G, T109I, A110S, N112Y, S113T, S114A, V115A, T116F, T121D, N121I,R123G, R123S, R123T, R123V, R123Y, R127H, R127K, R127E, R127S, R127Y,N145D, N145T, R159A, R159C, R159K, R159L, R159S, R159Y, T160E, T163D,N170L, R179L, T182V, T183E, T183I, and S185N, wherein the substitutionsare made at positions equivalent to the positions in a Cellulomonas 69B4protease comprising the amino acid sequence set forth in SEQ ID NO:8. Insome yet additional embodiments, the serine protease variant comprisesat least two substitutions selected from D2Q, V3L, N7L, I11A, I11Q,R14I, R14M, R16L, R16Q, N24A, N24E, N24H, N24M, N24Q, N24T, N24V, R35F,R35L, T36D, T36G, T36H, T36I, T36L, T36N, T36P, T36S, T36W, T36Y, A38L,A38R, A38S, A48Q, G49A, G54D, G54I, G54Q, G54N, R61V, A64F, A64H, A64Y,G65L, G65P, G65Q, G65S, G65T, G65Y, V66H, N67A, N67G, N67L, N67S, N67V,N67Y, L69H, L69S, Q71I, N73T, N74G, Y75F, Y75G, Y75I, S76A, S76D, S76E,S76I, S76L, S76N, S76T, S76V, S76W, S76Y, G77T, G78D, R79G, R79P, Q81P,H85F, H85K, H85L, H85Q, H85R, P89D, S92A, A93T, A93S, S99A, S99D, S99N,S99T, S99W, T109E, N112E, S113A, S114G, T116F, T121D, R123F, R123I,R123L, R127A, R127F, R127G, R127H, R127K, R127L, R127Q, R127S, R127T,R127Y, A132V, P134E, A143N, N157D, R159D, R159E, R159F, R159H, R159K,R159N, R159Y, G161K, N170Y, R179V, I181Q, D184F, D184H, G186E, G186I,G186V, and S187P, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8.

In some additional embodiments, the amino acid sequence of the serineprotease variant comprises at least two substitutions selected from F1T,A8D, A8G, T10E, T10L, T10Q, I11L, I11S, I11T, G12D, G12Y, R14E, R14L,R14N, R14P, 515E, R16A, R16G, R16I, R16N, N24L, N24S, V31F, R35A, A38D,A38F, A38N, A38V, A38Y, T40V, A41N, N42H, G49F, G49H, G49S, S51Q, S51T,G54A, G54L, G54M, N55F, R61H, R61K, R61M, R61S, R61T, A64N, A64S, A64T,A64V, A64W, G65R, G65V, V66D, N67F, N67K, N67M, N67Q, N67T, L69W, A70G,A70P, Q71D, Q71F, Q71H, Q71L, Q71T, G77N, G775, G78A, G78N, R79D, V80H,V80L, H85T, H85Y, T86N, A88F, P89N, P89V, V90I, V90P, V90T S92G, A93D,A93E, S99G, L111D, L111E, N112D, N112G, N112L, N112Q, S113G, T121E,R123E, R123K, R123Q, L125V, P134G, S140A, L142V, A143S, N145D, V150L,R159A, R159C, R159L, R159V, T160E, G161E, T163D, T163I, N170D, N170L,R179D, R179E, R179K, R179N, R179T, T181H, T183I, D184R, D184L, D184Q,D184T, S185W, S185I, G186L, S187E, S187Q, and S188Q, wherein thesubstitutions are made at positions equivalent to the positions in aCellulomonas 69B4 protease comprising the amino acid sequence set forthin SEQ ID NO:8. In some still further embodiments, the amino acidsequence of the serine protease variant comprises at least twosubstitutions selected from A8R, A8S, A8T, A8V, R14E, R14L, R14M, R16Q,N24A, N24E, N24Q, N24T, R35F, R35L, T36D, T36G, T36I, T36N, T36P, T36S,A38D, A38F, A38L, A38R, A38S, S51A, S51D, G54D, G54I, N55E, N55F, N55S,R61M, R61T, G63V, A64H, A64N, A64S, G65Q, G65P, G65R, G65S, G65T, G65Y,V66D, N67D, S76E, N67F, N67G, N67L, N67M, N67S, N67T, N67V, N67Y, L69H,L69S, L69V, L69W, N73T, N74G, Y75F, Y75G, S76C, S76D, S76I, S76L, S76N,S76W, S76Y, S76V, G77T, G78D, R79C, R79D, R79E, R79G, R79P, Q81V, A83N,T85A, H85Q, T86F, T86I, T86L, V90I, V90N, V90P, V90S, V90T, A93D, A93E,T107M, T107N, T107S, T109A, T109E, T109I, N112E, T121D, T121E, R123D,R123E, R123F, R123I, I126L, R127A, R127H, R127K, R127L, R127Q, R127S,R127Y, P134A, P134E, L142V, A143N, N145E, N145S, R150Y, R159C, R159D,R159E, R159F, R159K, R159Q, G161E, T163D, N170Y, I172V, G177M, R179A,R179D, R179E, R179I, R179K, R179L, R179M, R179N, R179T, R179V, R179Y,M180D, T182V, T183I, G186E, G186V, and S187P, wherein the substitutionsare made at positions equivalent to the positions in a Cellulomonas 69B4protease comprising the amino acid sequence set forth in SEQ ID NO:8. Insome yet additional embodiments, the amino acid sequence of the serineprotease variant comprises at least two substitutions selected from V3L,I4M, A8E, A8H, A8L, A8N, A8P, I11T, R14I, R14Q, R16L, N24H, N24L, N24M,N24V, R35A, R35E, T36V, T36Y, A38H, A38I, A38N, T40V, A41N, G49A, G49L,S51F, S51Q, G54A, G54E, G54M, G54Q, N55Q, N55V, R61V, T62I, G63D, G63L,G63P, G63Q, A64F, A64I, A64L, A64M, A64Q, A64R, A64T, A64V, A64W, G65A,G65D, V66E, N67A, N67C, N67Q, N67R, L69Q, A70G, A70P, A70S, Q71D, Q71M,S76A, S76Q, S76T, G77N, G77Q, R79L, Q81E, Q81H, Q81I, A83D, A83I, H85L,H85R, T86E, T86M, A88F, V90L, S92C, S92G, A93Q, R96K, T101S, W103M,W103Y, T107A, T107E, T107H, T107Q, T107V, T109G, T109H, T109L, T109N,A110S, A110T, N112D, S114G, T116F, T121L, R123A, R123H, R123K, R123L,R123P, R123Q, L125V, R127F, R127T, T129G, T129S, A132V, P134D, P134G,S140A, N145G, N145P, N145Q, N145T, Q146D, T151V, R159A, R159H, R159L,R159N, R159V, S161K, F166Y, N170C, N170D, P171M, A175T, A175V, Y176L,R179W, T182W, T183E, T183K, T183L, T183Q, G186I, G186L, G186P, G186T,S187E, S187T, and S188E, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8. In some additionalembodiments, the amino acid sequence of the serine protease variantcomprises at least two substitutions selected from: T10A, T10G, T10L,I11A, I11S, I11T, G12I, R14G, R14M, S15E, S15F, S15G, R16K, R16N, A22V,N24A, N24E, N24L, N24Q, N24T, N24V, G34A, T36G, T36I, T36N, T36S, A38F,A38T, G49A, G49F, S51A, G65V, L69H, L69S, Q71I, N73T, N74G, S76D, S76L,S76V, S76W, S76Y, G77T, V80A, V90I, V90P, S99N, S99V, T107K, T107R,N112S, S118A, E119R, R127F, P134D, P134E, P134H, P134L, P134R, P134V,S140A, L142V, V150L, 159F, R159K, T163I, F166Y, Q167N, N170Y, R179V,T182V, G186E, G186S, and G186V, wherein the substitutions are made atpositions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8.

The present invention also provides serine protease variants that haveimproved stability as compared to wild-type Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8. In someembodiments, the variants have improved thermostability as compared towild-type Cellulomonas 69B4 protease comprising the amino acid sequenceset forth in SEQ ID NO:8. In some particularly preferred embodiments,the variants comprise multiple substitutions selected fromT121E/R123F/R159E, R79T/R127Q/R179Q, R16Q/R79T/R127Q,R16Q/R79T/R123L/R159Q/R179Q, R16Q/R79T/R123L, R16Q/R79T,R16Q/R123L/R159Q, R14Q/T121E, R14L/R79T, R123L/R159Q, R123L/R127Q/R159Q,G12D/S15E/R35D/R123F/R159E, G12D/S15E/R159E, G12D/S15E,G12D/R35H/T121E/R123Q, G12D/R35H/R123Q, G12D/R35H/R123F/R159E,G12D/R35H, G12D/R35E/R123Q, G12D/R35E, G12D/R159E, G12D/R14Q/S15E/R35D,G12D/R14Q/R35H, G12D/R14Q/R159E, G12D/R14E, G12D/R127Q/R159E,G12D/R123E/R159E, and R35E/R123L/R127Q/R175Q, wherein the substitutionsare made at positions equivalent to the positions in a Cellulomonas 69B4protease comprising the amino acid sequence set forth in SEQ ID NO:8. Insome alternative embodiments, the variants have improved LAS stabilityas compared to wild-type Cellulomonas 69B4 protease comprising the aminoacid sequence set forth in SEQ ID NO:8. In some particularly preferredembodiments, the variants comprise multiple substitutions selected fromT121E/R123F/R159E, S15E/T121E/R123Q, S15E/R35H/R159E, S15E/R35E/R159E,S15E/R35E/R127Q/R159E, S15E/R35E, S15E/R35D/T121E/R123Q,S15E/R35D/R123Q, S15E/R35D/R123F/R159E, S15E/R35D, S15E/R159E,S15E/R127Q, S15E/R123Q, S15E/R123E, R79T/R127Q/R179Q, R35H/R159E,R35H/R127Q/R159E, R35H/R123D/R159E, R35F/R61S/R159Q, R35F/R159Q,R35E/T121E/R123E, R35E/R159E, R35E/R127Q, R35D/R159E, R35D/R127Q/R159E,R35D/R127Q, R35D/R123Q/R159E, R16Q/R79T/R159Q/R179Q, R16Q/R79T/R127Q,R16Q/R79T/R123L/R159Q/R179Q, R16Q/R79T/R123L/R159Q, R16Q/R79T/R123L,R16Q/R79T, R16Q/R61S/R159Q/R179Q, R16Q/R61S/R123L/R159Q,R16Q/R35F/R61S/R159Q, R16Q/R35F/R159Q, R16Q/R35F/R123L/R159Q, R16Q/R35F,R16Q/R159Q/R179Q, R16Q/R159Q, R16Q/R127Q/R179Q, R16Q/R127Q/R159Q,R16Q/R123L/R159Q, R14Q/T121E, R14Q/R35E/T121E, R14Q/R35E/R159E,R14Q/R35E, R14Q/R35D/R127Q, R14Q/R35D/R123E/R159E,R14Q/R35D/R123D/R159E, R14Q/R35D, R14Q/R123Q, R14L/R79T/R127Q/R159Q,R14L/R79T, R14L/R61S/R79T/R123L, R14L/R61S/R123L,R14L/R35F/R79T/R123L/R159Q, R14L/R35F/R61S, R14L/R127Q/R159Q/R179Q,R14L/R123L/R159Q, R14I/R35E/T121E/R159E, R14I/R35E/R127Q,R14I/R35E/R123E, R14I/R35D/R159E, R14I/R35D/R127Q/R159E, R14E/S15E/R35H,R14E/R35H/R127Q, R14D/S15E/R35E/R159E, R14D/R35H/R123Q/R159E,R127Q/R159E, R127Q/R159Q, R123Q/R159E, R123Q/R127Q/R159E, R123L/R159Q,R123L/R127Q/R159Q, R123F/R159E, R123E/R127Q/R159E, R123E/R127Q,G12D/S15E/R35H/R159E, G12D/S15E/R35H/R123F/R127Q/R159E,G12D/S15E/R35E/R159E, G12D/S15E/R35D/R127Q, G12D/S15E/R35D/R123F/R159E,G12D/S15E/R35D/R123E, G12D/S15E/R35D, G12D/S15E/R159E, G12D/S15E,G12D/R35H/T121E/R123Q, G12D/R35H/R159E, G12D/R35H/R123Q/R159E,G12D/R35H/R123Q, G12D/R35H/R123F/R159E, G12D/R35H, G12D/R35E/R159E,G12D/R35E/R123Q/R159E, G12D/R35E/R123Q, G12D/R35E, G12D/R35D/R159E,G12D/R35D/R127Q, G12D/R35D/R123Q/R159E, G12D/R35D, G12D/R159E,G12D/R14Q/S15E/R35D, G12D/R14Q/R35H, G12D/R14Q/R35E/R127Q/R159E,G12D/R14Q/R35D/R123H, G12D/R14Q/R159E, G12D/R14I/R35H, G12D/R14E,G12D/R14D/R35H/R123D/R127Q, G12D/R127Q/R159E, G12D/R123E/R159E,R127A/R159K, R14I/G65Q, R14I/G65Q/N67L/R159K,R14I/G65Q/N67L/Y75G/R127A/R159K, R14I/G65Q/R159K,R14I/G65Q/S76V/R127A/R159K, R14I/R127A, R14I/R127A/R159K, R14I/R159K,R14I/R35F, R14I/R35F/G65Q, R14I/R35F/G65Q/R127A/R159K,R14I/R35F/N67L/R127A/R159K, R14I/R35F/R127A/R159K, R14I/R35F/R159K,R14I/S76V, R14I/T36S/G65Q/R127A/R159K, R35F/R127A/R159K,R35F/S76A/R127A, 024A/G049A/A093H/S099N/R127K/A143N/R159K/I181Q,N024A/S076A/A093H/S099G/R127K/R159K,N024A/S076T/A093S/S099G/R127K/R159K,N024E/G049A/A093G/S099G/R127K/A143N/R159K/I181T,N024E/G049A/A093H//R127K/A143N/R159K/I181Q,N024E/G049A/A093H/S099A/R127K/A143N/R159K/I181T/V090I,N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q,N024H/G049A/A093T/S099A/R127K/A143N/R159K/I181Q,N024H/S076A/A093G/S099G/R127K/R159K,N024H/S076A/A093H/S099G/R127K/R159K,N024H/S076A/A093S/S099A/R127K/R159K/G054H/L069H,N024H/S076A/A093T/S099G/R127K/R159K,N024H/S076N/A093Q/S099W/R127K/R159K,N024H/S076V/A093Q/S099G/R127K/R159K,N024L/G049A/A093H/S099A/R127K/A143N/R159K/I181Q,N024L/G049A/A093S/S099A/R127K/A143N/R159K/I181Q,N024L/S076V/A093H/S099G/R127K, N024L/S076V/A093S/S099A/R127K/R159K,N024M/G049A/A093G/S099A/R127K/A143N/R159K/I181Q,N024M/G049A/A093H/S099D/R127K/A143N/R159K/I181Q,N024M/G049A/A093S/S099A/R127K/A143N/R159K/I181Q,N024M/G049A/A093S/S099W/R127K/A143N/R159K/I181Q,N024Q/G049A/A093H/S099A/R127K/A143N/R159K/I181T,N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181Q,N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T,N024Q/S076A/A093H/S099A/R127K/R159K/T039N,N024Q/S076A/A093H/S099W/R127K/R159K, N024Q/S076I/A093T/S099G/R159K,N024Q/S076T/A093S/R127K/R159K,N024S/S076A/A093G/S099G/R127K/R159K/G054A,N024S/S076A/A093H/S099T/R127K/R159K,N024S/S076A/A093S/S099W/R127K/R159K, N024S/S076A/A093T/S099W/R127K,N024S/S076T/A093Q/S099W/R127K/R159K,N024S/S076Y/A093T/S099A/R127K/R159K,N024T/G049A/A093G/S099A/R127K/A143N/R159K/I181Q,N024T/G049A/A093H/S099A/R127K/A143N/R159K/I181Q,N024T/G049A/A093H/S099A/R127K/A143N/R159K/I181T,N024T/G049A/A093H/S099D/R127K/A143N/R159K/I181Q,N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T,N024T/G049A/A093T/S099A/R127K/A143N/R159K/I181T,N024T/S076N/A093Q/S099T/R127K/R159K,N024T/S076T/A093T/S099N/R127K/R159K, N024V/S076A/R127K/R159K,N024V/S076V/A093Q/S099G/R127K/R159K, N024W/A093G/S099W/R127K/R159K,N024W/G049A/A093H/S099A/R127K/A143N/R159K/I181Q,N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181K,N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181Q,N024W/S076A/A093T/S099A/R127K/R159K,N024W/S076I/A093Q/S099G/R127K/R159K, N024W/S076N/A093T/S099G/R159K,N024W/S076T/A093H/S099A/R127K/R159K,N024W/S076T/A093H/S099W/R127K/R159K,N024W/S076V/A093H/S099A/R127K/R159K, N024W/S099W/R127K/R159K,N24A/G54E/S76D/A93G/R127K/R159K, N24E/A93G/R127K/R159K,N24E/G54L/S76E/A93G/R127K/R159K, N24E/G54Q/A93S/R127K/R159K,N24E/S76D/A93T/R127K/R159K, N24H/A93H/R127K/R159K,N24H/G54E/A93G/R127K/R159K, N24H/S76D/A93H/R127K/R159K,N24L/A93G/R127K/R159K, N24L/G54E/A93G/R127K/R159K,N24L/G54L/A93G/R127K/R159K, N24L/G54Q/S76A/A93H/R127K/R159K,N24L/S76T/A93G/R127K/R159K, N24L/S76T/A93H/R127K/R159K,N24M/A93G/R127K/R159K, N24M/A93H/R127K/R159K, N24M/A93S/R127K/R159K,N24M/A93T/R127K/R159K, N24M/G54E/A93H/R127K/R159K,N24M/G54E/S76N/A93S/R127K/R159K, N24M/G54I/A93H/R127K/R159K/S187I,N24Q/A93G/R127K/R159K, N24Q/G54D/A93H/R127K/R159K,N24Q/G54I/A93G/R127K/R159K, N24Q/G54I/S76E/A93H/R127K/R159K,N24Q/G54Q/A93G/R127K/R159K, N24Q/G54Q/S76T/A93H/R127K/R159K,N24Q/S76A/A93G/R127K/R159K, N24T/G54D/S76V/A93G/R127K/R159K,N24T/G54E/S76V/A93H/R127K/R159K, N24T/G54I/A93G/R127K/R159K,N24T/G54N/A93H/R127K/R159K, N24T/G54Q/S76N/A93G/R127K/R159K,N24T/G54Q/S76V/R127K/R159K, N24T/S76I/R127K/R159K,N24T/S76L/A93G/R127K/R159K, N24W/A93G/R127K/R159K,N24W/G54D/A93H/R127K/R159K, N24W/G54I/S76A/A93H/R127K/R159K,N24W/S76A/A93H/R127K/R159K, N24W/S76E/A93G/R127K/R159K,R014I/S076A/A093G/R127K/R159K/I181T,R014I/S076A/A093H/R127K/R159K/I181K,R014I/S076A/A093H/R127K/R159K/I181Q,R014I/S076A/A093H/R127K/R159K/I181T,R014I/S076D/A093H/R127K/R159K/I181Q,R014I/S076D/A093S/R127K/R159K/I181T,R014I/S076E/A093S/R127K/R159K/I181Q,R014I/S076E/A093T/R127K/R159K/I181K,R014I/S076I/A093S/R127K/R159K/I181Q,R014I/S076N/A093H/R127K/R159K/I181Q,R014I/S076T/A093G/R127K/R159K/I181Q,R014K/S076A/A093G/R127K/R159K/I181K,R014K/S076E/A093H/R127K/R159K/I181K,R014K/S076T/A093H/R127K/R159K/I181Q, R014L/S076A/A093H/R127K/R159K,R014L/S076A/A093H/R127K/R159K/I181Q,R014L/S076D/A093H/R127K/R159K/I181T,R014L/S076E/A093H/R127K/R159K/I181K,R014M/S076A/A093G/R127K/R159K/I181K,R014M/S076A/A093G/R127K/R159K/I181T,R014M/S076A/A093H/R127K/R159K/I181T,R014M/S076A/A093S/R127K/R159K/I181K,R014M/S076A/A093S/R127K/R159K/I181T,R014M/S076A/A093T/R127K/R159K/I181Q,R014M/S076D/A093S/R127K/R159K/I181T,R014M/S076E/A093G/R127K/R159K/I181T,R014M/S076E/A093H/R127K/R159K/I181T,R014M/S076E/A093S/R127K/R159K/I181T,R014M/S076N/A093G/R127K/R159K/I181K,R014M/S076N/A093G/R127K/R159K/I181T,R014M/S076N/A093H/R127K/R159K/I181Q,R014M/S076N/A093H/R127K/R159K/I181T,R014M/S076N/A093S/R127K/R159K/I181T,R014M/S076N/A093T/R127K/R159K/I181T,R014M/S076T/A093H/R127K/R159K/I181K,R014M/S076V/A093G/R127K/R159K/I181Q,R014M/S076V/A093H/R127K/R159K/I181Q, G54E/R14L, G54L/R127S,N24D/G54F/R127C, N24E/R127S, N24E/R159C, N24G/G54I/R127S,N24H/R159Y/T46I, N24I/R127V/R14V, N24T/R127Q/R179F, R127A/R159V/R179F,R127c/R14W, R127S/R159N/R123L, R14A/N24F/R159L, R14A/R127L,R14A/R127Y/R159W, R14A/R159W, R14c/S114F/R159G, R14F/R127L/R159F,R14F/R127Q/R159W, R14F/R127S/R159V, R14F/R127V/R159F, R14G/N24L/R159G,R14G/N24S/R127C, R14G/R127c/G63E, R14G/R127G, R14G/R127P,R14L/N24S/R159F, R14L/N24V/R127S/R159I, R14L/R123L, R14L/R127c/R159G,R14L/R127S, R14L/R127S/R159G, R14L/R127V, R14L/R127V/R159F,R14L/R127W/R123Y, R14L/R127Y, R14L/R127Y/R159F, R14L/R159G, R14L/R159L,R14L/R159S, R14L/R159V, R14L/R159W, R14M/N24L/R159S/R123V, R14M/R159F,R14Q/R123F, R14S/N24E/R127W, R14S/N24L/R159G, R14S/R127L/R159F,R14S/R127V, R14T/R14P/R159F, R14T/N24A, R14T/N24T/R127Q,R14T/N24T/R127Y/R159W, R14T/R127Y, R14V/N24A/R127I/R159A,R14V/N24D/R127C, R14V/N24G/P189S, R14V/N24S, R14V/N24Y/R127S/R159G,R14V/R127A, R14V/R127c/R159S, R14V/R127M/R159V, R14V/R127S/R159G,R14V/R127T/R159Y, R14V/R127V, R14V/R159F, R14V/R159V, R14V/R159W,R14W/N24T/R123E, R14W/R123L, R14W/R123V, R14W/R127Q/R159W, R14W/R159V,R159V/G49D, G012D/R035E/G065E, G012D/R035E/G065E, G012D/R035E/Q081P,G012D/R035E/R016S/A064T, G012D/R035E/R159W, G012D/R035E/R179I,G012D/R035E/S092T/I181V, R14I/N24A/A64K/R123F/R159E/D184T,R14I/A64K/R123F/R159F/D184T, R14I/A64K/R123F/R159E/D184T,R14I/N24Q/R35E/A64K/R123F/D184T, R14I/N24Q/A64K/N67S/R123F/R159F/D184T,R14I/N24A/R35E/A64K/N67S/R123F/R159E/D184T,R14I/N24A/R35E/A64K/N67S/G78D/R123F/D184T,R14I/N24A/R35D/A64K/G78D/R123F/R127K/R159E/D184T,R14I/N24A/R35D/A64K/R123F/R127K/R159F/D184T,R14I/N24T/R35D/A64K/G78D/R123F/R127Q/R159F/D184T, R14I/A64K/R123F/D184T,R14I/N24A/A64K/R123F/R159N/D184T,R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T,R14I/N24E/R35E/A64K/G78D/R123F/R127Q/R159F/D184T,R14I/N24E/A64K/R123F/R127K/R159K/D184T, R14I/N24A/A64K/R123F/D184T,R14I/A64K/R123F/R127K/R159F/D184T, R14I/A64K/R123F/R159E/D184T,R14I/A64K/R123F/R159N/D184T, R14I/A64K/R123F/R159K/D184T,R14I/A64K/R123F/R127Y/R159E/D184T,R14I/N24A/R35E/A64K/N67A/G78D/R123F/D184T,R14I/A64K/R123F/R127Y/R159K/D184T,R14I/N24Q/A64K/R123F/R127Q/R159K/D184T, R14I/A64K/R123F/R159K/D184T,R14I/N24Q/A64K/G78D/R123F/R127Q/R159N/D184T,R14I/N24E/A64K/N67L/G78D/R123F/R159K/D184T,R14I/N24A/R35E/A64K/G78D/R123F/R127K/R159E/D184T,R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T,R14I/N24A/R35D/A64K/N67A/R123F/R159F/D184T,R14I/N24E/R35E/G54D/A64K/N67L/G78D/R123F/R127K/D184T,R14I/A64K/G78D/R123F/R127Q/R159N/D184T,R14I/N24A/R35E/A64K/G78D/R123F/R159N/D184T,R14I/A64K/R123F/R127K/R159E/D184T,R14I/N24A/R35E/A64K/N67S/G78D/R123F/R127K/R159F/D184T,R14I/A64K/G78D/R123F/R159E/D184T,R14I/N24E/R35D/A64K/N67A/G78D/R123F/R159K/D184T, N24T/R35D/G78D/R159K,N24T/R35E/N67A/G78D/R127Q, N24Q/R35E, R127K/R159N, R35D/R159E,R35E/G54D/N67S/G78D/R159K, N24Q/G54D/G78D/R159N, R127K/R159E,R127Q/R159K, N24E/R35E/G54D/N67S/R127K/R159N, R35D/G78D/R159K,N67S/R159E, G54D/R127K/R159K, G78D/R127K/R159K, G78D/R127K/R159E,N24E/R35D/G78D/R127K/R159N, R35D/G78D/R127K/R159N, N24A/R35E/G78D/R159N,N24Q/R35D/N67S/R127K/R159E, N24T/R35D/G78D/R159K, N67S/G78D/R127K/R159K,N24Q/R35D/R127K/R159K, N24E/G54D/G78D/R159K, R35D/R159K, R35E/R159K,R127K/R159K, R35E/N67S/G78D/R127Q, N24E/R35D/G78D,R35D/G78D/R127K/R159E, N24E/R35E/G54D/N67S/G78D/R127K/R159K,N24T/N67S/R159E, N24D/R35D/G78D/R159F, N24Q/R35D/N67S/G78D/R127K/R159F,R35D/G78D/R127Q/R159K, G78D/R159F, N24A/N67S/R159K, G78D/R127Q/R159K,N24T/G54D/N67S/G78D/R127Y/R159E, R14I/A63K/G78D/R123F/D184T,R14I/A63K/R123F/R159E/D184T, R14I/A63K/R123F/R159F/D184T,R14I/A63K/R123F/R159K/D184T, R14I/A63K/R123F/R159N/D184T,R14I/A63K/R123K/D184T, R14I/A63K/R123Q/D184T, R14I/A63K/R123Y/D184T,R14I/A64K/G78D/T86K/T116E/R123F, R14I/A64K/T86K/T116E/R123F/R159E,R14I/A64K/T86K/T116E/R123F/R159K, R14I/A64K/T86K/T116E/R123K,R14I/A64K/T86K/T116E/R123Q, R14I/A64K/T86K/T116E/R123Y,R14I/G54D/A63K/R123F/D184T, R14I/G54D/A64K/T86K/T116E/R123F,R141/G54D/S76N/A93H/R127K/R159K/I181Q,R14I/G54D/S76V/A93S/R127K/R159K/I181K, R14I/N24A/A63K/R123F/D184T,R14I/N24A/A64K/T86K/T116E/R123F, R14I/N24E/A63K/R123F/D184T,R14I/N24E/A64K/T86K/T116E/R123F, R14I/N24Q/A63K/R123F/D184T,R14I/N24Q/A64K/T86K/T116E/R123F, R14I/N24T/A63K/R123F/D184T,R14I/N24T/A64K/T86K/T116E/R123F, R14I/N24TS76N/A93H/R127K/R159K/I181Q,R14I/N24TS76V/A93S/R127K/R159K/I181K,R14I/N67AS76N/A93H/R127K/R159K/I181Q,R14I/N67LS76N/A93H/R127K/R159K/I181Q,R14I/N67SS76N/A93H/R127K/R159K/I181Q, R14I/R35D/A64K/T86K/T116E/R123F,R14I/R35D/S76N/A93H/R127K/R159K/I181Q, R14I/R35E/A63K/R123F/D184T,R14I/R35E/A64K/T86K/T116E/R123F, R14I/R35E/S76N/A93H/R127K/R159K/I181Q,R14I/R35E/S76V/A93S/R127K/R159K/I181K, R14I/R35K/A63K/R123F/D184T,R14I/S76N/A93H/R127K/R159E/I181Q, R14I/S76N/A93H/R127K/R159F/I181Q,R14I/S76N/A93H/R127K/R159N/I181Q, R14I/S76N/A93H/R127Q/R159K/I181Q,R14I/S76N/A93H/R127Y/R159K/I181Q, R14I/S76N/G78D/A93H/R127K/R159K/I181Q,R14I/S76V/A93S/R127K/R159F/I181K, R14I/S76V/A93S/R127K/R159N/I181K,R14I/S76V/A93S/R127Q/R159K/I181K, R14I/S76V/A93S/R127Y/R159K/I181K,R14M/G54D/S76N/A93G/R127K/R159K/I181K,R14M/N24A/S76N/A93G/R127K/R159K/I181K,R14M/N24E/S76N/A93G/R127K/R159K/I181K,R14M/N24Q/S76N/A93G/R127K/R159K/I181K,R14M/N24T/S76N/A93G/R127K/R159K/I181K,R14M/N67S/S76N/A93G/R127K/R159K/I181K,R14M/R35D/S76N/A93G/R127K/R159K/I181K,R14M/R35E/S76N/A93G/R127K/R159K/I181K, R14M/S76N/A93G/R127K/R159E/I181K,R14M/S76N/A93G/R127K/R159F/I181K, R14M/S76N/A93G/R127K/R159N/I181K,R14M/S76N/A93G/R127Q/R159K/I181K, R14M/S76N/A93G/R127Y/R159K/I181K, andR14M/S76N/G78D/A93G/R127K/R159K/I181K, wherein the substitutions aremade at positions equivalent to the positions in a Cellulomonas 69B4protease comprising the amino acid sequence set forth in SEQ ID NO:8.

The present invention also provides serine protease variants that haveimproved activity, as compared to wild-type Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8. In somepreferred embodiments, the variants have improved caseinolytic activityas compared to wild-type Cellulomonas 69B4 protease comprising the aminoacid sequence set forth in SEQ ID NO:8. In some particularly preferredembodiments, the variants comprise multiple substitutions selected fromR14L/R79T, G12D/R35H/R159E, G12D/R35H/R123Q, G12D/R35H/R123F/R159E,G12D/R35H, G12D/R35E/R159E, G12D/R35E/R123Q/R159E, G12D/R35E/R123Q,G12D/R35E, G12D/R35D/R159E, G12D/R35D/R123Q/R159E, G12D/R35D,G12D/R159E, G12D/R14Q/R35H, G12D/R14I/R35H,024E/G049A/A093H/R127K/A143N/R159K/I181Q, N24M/S76V/A93H/R127K/R159K,R14I/N24E/R35D/A64K/N67A/G78D/R123F/R159K/D184T R127A/R159K, R14I/G65Q,and R14I/G65Q/R159K R14I/S76V, wherein the substitutions are made atpositions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8. In somealternative embodiments, the variants have improved keratinolyticactivity as compared to wild-type Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8. In some particularlypreferred embodiments, the variants comprise multiple substitutionsselected from N024E/G049A/A093H/R127K/A143N/R159K/I181Q,N024E/G049A/A093H/S099A/R127K/A143N/R159K/I181T/V090I,N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q,N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181Q,N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T,N024Q/G049A/A093S/S099N/R127K/A143N/R159K/I181Q,N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T,N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181Q,N24A/G54E/S76D/A93G/R127K/R159K, N24E/A93G/R127K/R159K,N24E/G54L/S76E/A93G/R127K/R159K, N24E/G54Q/A93S/R127K/R159K,N24E/S76D/A93T/R127K/R159K, N24M/G54E/A93H/R127K/R159K,N24M/G54E/S76N/A93S/R127K/R159K, N24Q/A93G/R127K/R159K,N24Q/G54D/S76L/A93G/R127K/R159K, N24Q/G54I/S76E/A93H/R127K/R159K,N24T/G54D/S76V/A93G/R127K/R159K, N24T/G54E/S76V/A93H/R127K/R159K,N24W/G54D/A93H/R127K/R159K, N24W/S76E/A93G/R127K/R159K,R014I/S076A/A093G/R127K/R159K/I181T,R014I/S076A/A093H/R127K/R159K/I181Q,R014I/S076D/A093H/R127K/R159K/I181Q,R014I/S076D/A093H/R127K/R159K/I181T,R014I/S076D/A093S/R127K/R159K/I181T,R014I/S076E/A093S/R127K/R159K/I181Q,R014I/S076E/A093T/R127K/R159K/I181K,R014I/S076I/A093S/R127K/R159K/I181Q,R014I/S076N/A093H/R127K/R159K/I181Q,R014I/S076T/A093G/R127K/R159K/I181Q,R014I/S076V/A093H/R127K/R159K/I181Q,R014K/S076A/A093S/R127K/R159K/I181T,R014K/S076E/A093H/R127K/R159K/I181T,R014K/S076E/A093S/R127K/R159K/I181T,R014K/S076T/A093H/R127K/R159K/I181Q, R014L/S076A/A093H/R127K/R159K,R014L/S076A/A093H/R127K/R159K/I181Q,R014L/S076D/A093H/R127K/R159K/I181T,R014L/S076E/A093H/R127K/R159K/I181K,R014M/S076A/A093G/R127K/R159K/I181T,R014M/S076A/A093H/R127K/R159K/I181T,R014M/S076A/A093S/R127K/R159K/I181K,R014M/S076A/A093S/R127K/R159K/I181T,R014M/S076A/A093T/R127K/R159K/I181Q,R014M/S076D/A093S/R127K/R159K/I181T,R014M/S076E/A093G/R127K/R159K/I181T,R014M/S076E/A093H/R127K/R159K/I181T,R014M/S076E/A093S/R127K/R159K/I181T,R014M/S076N/A093G/R127K/R159K/I181K,R014M/S076N/A093G/R127K/R159K/I181T,R014M/S076N/A093H/R127K/R159K/I181T,R014M/S076N/A093S/R127K/R159K/I181T,R014M/S076V/A093G/R127K/R159K/I181Q,R14I/N24Q/A64K/G78D/R123F/R159K/D184T,R14I/N24A/A64K/N67S/G78D/R123F/R159K/D184T,R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T,R14I/N24E/A64K/R123F/R127K/R159K/D184T,R14I/N24Q/A64K/R123F/R127Q/R159K/D184T,R14I/N24Q/A64K/G78D/R123F/R127Q/R159N/D184T,R14I/N24E/A64K/N67L/G78D/R123F/R159K/D184T,R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T, R127Q/R159K,G78D/R127K/R159K, N67S/G78D/R127K/R159K, R35D/R159K, G78D/R127Q/R159K,N24A/N67A/R159K, T36S/R127Q/R159E, S15E/T121E/R123Q, S15E/R35H/R159E,S15E/R35E, S15E/R159E, S15E/R123Q, S15E/R123E, R79T/R127Q/R179Q,R35H/R159E, R35H/R127Q/R159E, R35F/R61S/R159Q, R35F/R159Q, R35E/R159E,R35E/R127Q, R35D/R159E, R35D/R127Q, R16Q/R79T/R159Q/R179Q,R16Q/R79T/R127Q, R16Q/R79T/R123L, R16Q/R79T, R16Q/R35F/R123L/R159Q,R16Q/R159Q/R179Q, R16Q/R127Q/R159Q, R16Q/R123L/R159Q, R14Q/T121E,R14Q/R35E/T121E, R14Q/R35E/R159E, R14Q/R35E, R14Q/R35D/R127Q, R14Q/R35D,R14L/R79T/R127Q/R159Q, R14L/R61S/R79T/R123L, R14L/R35F/R61S,R14L/R127Q/R159Q/R179Q, R14L/R123L/R159Q, R14I/R35E/R127Q,R14I/R35E/R123E, R14I/R35D/R159E, R14E/R35H/R127Q, R127Q/R159E,R127Q/R159Q, R123Q/R159E, R123Q/R127Q/R159E, R123L/R159Q,R123L/R127Q/R159Q, R123F/R159E, R123E/R127Q/R159E, R123E/R127Q,G12D/S15E/R35H/R159E, G12D/S15E/R35D, G12D/S15E/R159E, G12D/S15E,G12D/R35H/T121E/R123Q, G12D/R35H/R159E, G12D/R35H/R127Q/R159E,G12D/R35H/R123Q, G12D/R35H/R123F/R159E, G12D/R35H, G12D/R35E/R159E,G12D/R35E/R123Q/R159E, G12D/R35E/R123Q, G12D/R35E, G12D/R35D/R159E,G12D/R35D/R127Q, G12D/R35D, G12D/R159E, G12D/R14Q/R35H,G12D/R14Q/R35D/R123H, G12D/R14Q/R159E, G12D/R14I/R35H, G12D/R14E,G12D/R127Q/R159E, G12D/R123E/R159E, R127A/R159K, R14I/G65Q,R14I/G65Q/R127A/R159K, R14I/G65Q/R159K, R14I/R127A, R14I/R127A/R159K,R14I/R159K, R14I/R35F, R14I/R35F/G65Q, R14I/R35F/G65Q/R127A/R159K,R14I/R35F/R159K, R14I/S76V, R14I/T36S/G65Q/R127A/R159K, andR35F/R127A/R159K, wherein the substitutions are made at positionsequivalent to the positions in a Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8.

The present invention also provides serine protease variants that haveimproved wash performance activity as compared to wild-type Cellulomonas69B4 protease comprising the amino acid sequence set forth in SEQ IDNO:8. In some preferred embodiments, the variants comprise multiplesubstitutions selected from N24F/R159G,/N24F/R159L/R123V,N24I/R127S,/N24L/R159S, N24S/R159A, N24V/R159L, N24Y/R159F,R14A/N24K/R127S, R14A/R159W, R14L/N24Y, R14L/R159G, R14L/R159S,R14L/T109M, R14L/T39P, R14M/R159W, R14S/N24V, R14S/N24Y, R14T/N24A,R14V/N24G/P189S, R14V/R159W, R127A/R159K, R14I/G65Q, R14I/S76V,R14I/G65Q/N67L/R159K, R14I/G65Q/R159K, R14I/S76V, R35F/R159Q, R16Q/R79T,R16Q/R35F, R16Q/R159Q, R14L/R79T, R123L/R159Q, G12D/S15E, G12D/R35H,G12D/R35D, N024E/G049A/A093H//R127K/A143N/R159K/I181Q,N024H/S076A/A093S/S099A/R127K/R159K/G054H/L069H,N024H/S076A/A093T/S099G/R127K/R159K,N024L/S076V/A093S/S099A/R127K/R159K,N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T,N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T,N24E/G54Q/A93S/R127K/R159K, N24H/A93H/R127K/R159K,N24H/G54L/S76V/A93H/R127K/R159K, N24L/G54Q/S76A/A93H/R127K/R159K,N24M/A93G/R127K/R159K, N24M/G54E/A93H/R127K/R159K,N24M/S76V/A93H/R127K/R159K, N24Q/G54Q/S76T/A93H/R127K/R159K,N24Q/S76A/A93G/R127K/R159K, N24T/G54Q/S76N/A93G/R127K/R159K,N24T/S76I/R127K/R159K, N24W/G54D/A93H/R127K/R159K,N24W/S76A/A93H/R127K/R159K, N24W/S76T/A93G/R127K/R159K,N24W/S76V/A93G/R127K/R159K, N24W/S76Y/A93G/R127K/R159K,R014I/S076A/A093G/R127K/R159K/I181T,R014I/S076A/A093H/R127K/R159K/I181K,R014I/S076A/A093H/R127K/R159K/I181Q,R014I/S076A/A093H/R127K/R159K/I181T,R014I/S076D/A093S/R127K/R159K/I181T,R014I/S076E/A093T/R127K/R159K/I181K,R014I/S076N/A093H/R127K/R159K/I181Q,R014I/S076V/A093H/R127K/R159K/I181Q,R014I/S076V/A093S/R127K/R159K/I181K,R014K/S076A/A093S/R127K/R159K/I181T,R014K/S076E/A093H/R127K/R159K/I181K,R014K/S076I/A093S/R127K/R159K/I181T,R014K/S076T/A093H/R127K/R159K/I181K,R014K/S076T/A093H/R127K/R159K/I181T,R014K/S076V/A093H/R127K/R159K/I181K, R014L/S076A/A093H/R127K/R159K,R014L/S076A/A093H/R127K/R159K/I181Q,R014L/S076D/A093H/R127K/R159K/I181T,R014L/S076E/A093H/R127K/R159K/I181K,R014M/S076A/A093G/R127K/R159K/I181K,R014M/S076A/A093G/R127K/R159K/I181T,R014M/S076A/A093H/R127K/R159K/I181T,R014M/S076A/A093S/R127K/R159K/I181K,R014M/S076A/A093S/R127K/R159K/I181T,R014M/S076A/A093T/R127K/R159K/I181Q,R014M/S076I/A093H/R127K/R159K/I181T,R014M/S076I/A093S/R127K/R159K/I181T,R014M/S076N/A093G/R127K/R159K/I181K,R014M/S076N/A093G/R127K/R159K/I181T,R014M/S076N/A093H/R127K/R159K/I181T,R014M/S076N/A093S/R127K/R159K/I181T,R014M/S076T/A093H/R127K/R159K/I181K,R014M/S076V/A093G/R127K/R159K/I181Q,R014M/S076W/A093H/R127K/R159K/I181K,R014M/S076Y/A093H/R127K/R159K/I181K,R014M/S076Y/A093H/R127K/R159K/I181T, G012D/R035E/D184N,G012D/R035E/NO₆₇K, R14I/A64K/R123F/R159F/D184T,R14I/A64K/R123F/R159F/D184T, R14I/N24Q/A64K/G78D/R123F/R159K/D184T,R14I/N24Q/A64K/N67S/R123F/R159F/D184T,R14I/N24Q/A64K/N67A/R123F/R159K/D184T,R14I/N24A/A64K/N67S/G78D/R123F/R159K/D184T, R14I/A64K/R123F/D184T,R14I/N24A/A64K/R123F/R159N/D184T,R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T,R14I/N24Q/A64K/N67A/R123F/R159K/D184T,R14I/N24E/A64K/R123F/R127K/R159K/D184T, R14I/N24A/A64K/R123F/D184T,R14I/A64K/R123F/R127K/R159F/D184T, R14I/A64K/R123F/R159N/D184T,R14I/A64K/R123F/R159K/D184T, R141/A64K/R123F/R127Y/R159K/D184T,R14I/N24Q/A64K/R123F/R127Q/R159K/D184T, R14I/A64K/R123F/R159K/D184T,R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T,R14I/A64K/N67S/G78D/R123F/R127K/R159K/D184T,R14I/N24Q/A64K/N67A/R123F/R127K/R159K/D184T, R127K/R159K,N24A/N67S/R159K, N24A/N67A/R159K, R14I/A63K/G78D/R123F/D184T,R14I/A63K/N67A/R123F/D184T, R14I/A63K/N67L/R123F/D184T,R14I/A63K/N67S/R123F/D184T, R14I/A63K/R123F/R159F/D184T,R14I/A63K/R123F/R159K/D184T, R14I/A63K/R123F/R159N/D184T,R14I/A63K/R123K/D184T, R14I/A63K/R123Q/D184T, R14I/A63K/R123Y/D184T,R14I/A64K/G78D/T86K/T116E/R123F, R14I/A64K/N67A/T86K/T116E/R123F,R14I/A64K/N67L/T86K/T116E/R123F, R14I/A64K/T86K/T116E/R123F/R159K,R14I/A64K/T86K/T116E/R123K, R14I/G54D/A63K/R123F/D184T,R14I/G54D/A64K/T86K/T116E/R123F, R14I/N24A/A63K/R123F/D184T,R14I/N24A/A64K/T86K/T116E/R123F, R14I/N24A/S76V/A93S/R127K/R159K/I181K,R14I/N24E/A63K/R123F/D184T, R14I/N24E/A64K/T86K/T116E/R123F,R14I/N24Q/A63K/R123F/D184T, R14I/N24Q/A64K/T86K/T116E/R123F,R14I/N24Q/S76V/A93S/R127K/R159K/I181K, R14I/N24T/A63K/R123F/D184T,R14I/N24T/A64K/T86K/T116E/R123F, R14I/N24TS76V/A93S/R127K/R159K/I181K,R14I/N67SS76N/A93H/R127K/R159K/I181Q, R14I/R35E/A63K/R123F/D184T,R14I/R35K/A63K/R123F/D184T, R14I/S76V/A93S/R127K/R159F/I181K,R14I/S76V/A93S/R127K/R159N/I181K, R14I/S76V/A93S/R127Y/R159K/I181K,R14M/N24T/S76N/A93G/R127K/R159K/I181K,R14M/N67S/S76N/A93G/R127K/R159K/I181K, R14M/S76N/A93G/R127K/R159F/I181K,and R14M/S76N/G78D/A93G/R127K/R159K/I181K, wherein the substitutions aremade at positions equivalent to the positions in a Cellulomonas 69B4protease comprising the amino acid sequence set forth in SEQ ID NO:8. Insome embodiments, the variants have improved dishwashing performanceactivity as compared to wild-type Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8. In some preferredembodiments, the variants comprise multiple substitutions selected fromR14N/R127K/R159L, R14I/A64K/T86K/N112E/R123F/D184T,G12D/R35E/G63R/R79K/T109M, R14L, G12D/R35E, andR14M/S76D/A93H/R127K/R159K/I181K, wherein the substitutions are made atpositions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8. In somestill further embodiments, the variants have improved stain removalactivity as compared to wild-type Cellulomonas 69B4 protease comprisingthe amino acid sequence set forth in SEQ ID NO:8. In some preferredembodiments, the variants comprise multiple substitutions selected from:G54E/R14L, N24D/R127Y/R159V, N24E/R127S, N24E/R159C, N24F/R159G,N24F/R159G/G54E, N24F/R159L/R123V, N24G/R127Y, N24H/R159Y/T46I,N24I/R127S, N24I/R127V/R14/N24L/R159S, N24S/R159A, N24V/R127M/R159V,N24V/R127S/R159H, N24V/R159L, N24Y/G54A, N24Y/R127L, N24Y/R127S,N24Y/R127V, N24Y/R159F, R127A/R159F, R127H/R159Q, R127H/R159T/S185F,R127M/R159V, R127S/R159G, R127S/R159L, R127T/R159F, R127V/R159G,R127Y/R159L, R14A/N24K/R127S, R14A/R127Y/R159W, R14G/N24S/R127C,R14G/R127G, R14L/N24D, R14L/N24Y, R14L/R123L, R14L/R127S, R14L/R127V,R14L/R127Y, R14L/R159G, R14L/R159S, R14L/T39P, R14M/N24L/R159S/R123V,R14M/R159F, R14M/R159W, R14Q/R123F, R14S/N24E/R127W, R14S/N24V,R14S/N24Y, R14T/N24T/R127Q, R14T/R127Y, R14V/N24D/R127C,R14V/N24G/P189S, R14V/N24L/R127F, R14V/R127A, R14V/R159F, R14V/R159W,R14W/N24A, R14W/R123L, R14W/R123V, R14W/R159V, R159V/G49D, R159V/R123G,N024E/G049A/A093H/R127K/A143N/R159K/I181Q,N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q,N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T,N24A/G54E/S76D/A93G/R127K/R159K, N24E/A93G/R127K/R159K,N24E/G54L/S76E/A93G/R127K/R159K, N24E/G54Q/A93S/R127K/R159K,N24E/S76D/A93T/R127K/R159K, N24H/G54E/A93G/R127K/R159K,N24L/S76T/A93G/R127K/R159K, N24M/A93S/R127K/R159K,N24M/A93T/R127K/R159K, N24M/G54E/A93H/R127K/R159K,N24M/G54E/S76N/A93S/R127K/R159K, N24M/S76V/A93H/R127K/R159K,N24Q/A93G/R127K/R159K, N24Q/G54D/S76L/A93G/R127K/R159K,N24Q/G54I/S76T/A93G/R127K/R159K, N24Q/G54Q/A93G/R127K/R159K,N24Q/S76A/A93G/R127K/R159K, N24T/G54Q/S76N/A93G/R127K/R159K,N24T/G54Q/S76V/R127K/R159K, N24T/S76I/R127K/R159K,N24W/A93G/R127K/R159K, N24W/G54D/A93H/R127K/R159K,R014I/S076A/A093G/R127K/R159K/I181T,R014I/S076A/A093H/R127K/R159K/I181Q,R014I/S076D/A093H/R127K/R159K/I181Q,R014I/S076D/A093H/R127K/R159K/I181T,R014I/S076D/A093S/R127K/R159K/I181T,R014I/S076E/A093S/R127K/R159K/I181Q,R014I/S076E/A093T/R127K/R159K/I181K,R014I/S076N/A093H/R127K/R159K/I181Q, R014L/S076A/A093H/R127K/R159K,R014L/S076A/A093H/R127K/R159K/I181Q,R014L/S076D/A093H/R127K/R159K/I181T,R014M/S076A/A093G/R127K/R159K/I181T,R014M/S076A/A093S/R127K/R159K/I181K,R014M/S076A/A093S/R127K/R159K/I181T,R014M/S076A/A093T/R127K/R159K/I181Q,R014M/S076D/A093S/R127K/R159K/I181T,R014M/S076E/A093H/R127K/R159K/I181T,R014M/S076E/A093S/R127K/R159K/I181T,R014M/S076N/A093G/R127K/R159K/I181K,R014M/S076N/A093G/R127K/R159K/I181T,R014M/S076N/A093H/R127K/R159K/I181T,R014M/S076N/A093S/R127K/R159K/I181T, T36S/R127Q/R159E, S15E/R35E,S15E/R35D, S15E/R159E, S15E/R127Q, S15E/R123Q, S15E/R123E,R79T/R127Q/R179Q, R35H/R159E, R35F/R61S/R159Q, R35F/R159Q, R35D/R127Q,R16Q/R79T/R159Q/R179Q, R16Q/R79T/R123L, R16Q/R79T, R16Q/R35F,R16Q/R159Q/R179Q, R16Q/R159Q, R14Q/T121E, R14Q/R35E, R14Q/R35D,R14Q/R123Q, R14L/R79T, R127Q/R159E, R127Q/R159Q, R123Q/R159E,R123L/R159Q, R123F/R159E, G12D/S15E, G12D/R35H/R159E, G12D/R35H/R123Q,G12D/R35H, G12D/R35E, G12D/R35D, G12D/R159E, G12D/R14Q/R35H,G12D/R14I/R35H, G65Q/R127A/R159K, R127A/R159K, R14I/G65Q,R14I/G65Q/N67L/R159K, R14I/G65Q/R127A, R14I/G65Q/R127A/R159K,R14I/G65Q/R159K, R14I/R127A, R14I/R127A/R159K, R14I/R159K, R14I/R35F,R14I/R35F/G65Q, R14I/R35F/R159K, R14I/S76V, and R35F/R127A/R159K,wherein the substitutions are made at positions equivalent to thepositions in a Cellulomonas 69B4 protease comprising the amino acidsequence set forth in SEQ ID NO:8.

The present invention also provides serine protease variants that haveat least one altered surface property, as compared to wild-typeCellulomonas 69B4 protease comprising the amino acid sequence set forthin SEQ ID NO:8. In some preferred embodiments, the variants comprise atleast two substitutions at sites selected from the group consisting of1, 2, 4, 7, 8, 10, 11, 12, 13, 14, 15, 16, 22, 24, 25, 32, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 57, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 73, 74, 75, 76, 77,78, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 123, 124, 126, 127, 128, 130, 131,132, 133, 134, 135, 137, 143, 144, 145, 146, 147, 148, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, and184, wherein the substitutions are made at positions equivalent to thepositions in a Cellulomonas 69B4 protease comprising the amino acidsequence set forth in SEQ ID NO:8

In some particularly preferred embodiments, the variant proteases haveat least one improved property as compared to the wild-type Cellulomonas69B4 protease comprising the amino acid sequence set forth in SEQ IDNO:8. In some particularly preferred embodiments, the at least oneimproved property is selected from the group consisting of acidstability, thermostability, casein hydrolysis, keratin hydrolysis,cleaning performance, and LAS stability.

The present invention also provides expression vectors comprising apolynucleotide sequence encoding the serine protease variants having anamino acid sequence comprising at least two amino acid substitutions,wherein the substitutions are made at positions equivalent to positionsin a Cellulomonas 69B4 protease comprising the amino acid sequence setforth in SEQ ID NO:8. The present invention also provides host cellscomprising at least one expression vector. In some preferredembodiments, the host cell is a Bacillus sp., while in some otherembodiments, the host cell is a Streptomyces sp., in yet some otherembodiments, the host cell is an Aspergillus sp., and in some stillfurther embodiments, the host cell is a Trichoderma sp. The presentinvention also provides serine protease variants produced the hostcells.

The present invention also provides compositions comprising at least aportion of the serine protease variants having an amino acid sequencecomprising at least two amino acid substitutions, wherein thesubstitutions are made at positions equivalent to the positions in aCellulomonas 69B4 protease comprising the amino acid sequence set forthin SEQ ID NO:8, provided herein.

The present invention also further provides polynucleotide sequencesencoding the serine protease variants having amino acid sequencescomprising at least two amino acid substitutions, wherein thesubstitutions are made at positions equivalent to positions in aCellulomonas 69B4 protease comprising the amino acid sequence set forthin SEQ ID NO: 8. The present invention further provides expressionvectors comprising a polynucleotide sequence encoding the serineprotease variants having an amino acid sequence comprising at least twoamino acid substitutions, wherein the substitutions are made atpositions equivalent to positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8. The presentinvention also provides host cells comprising at least one expressionvector. In some preferred embodiments, the host cell is a Bacillus sp.,while in some other embodiments, the host cell is a Streptomyces sp., inyet some other embodiments, the host cell is an Aspergillus sp., and insome still further embodiments, the host cell is a Trichoderma sp. Thepresent invention also provides serine protease variants produced thehost cells.

The present invention further provides cleaning compositions comprisingserine protease variants having amino acid sequences comprising at leasttwo amino acid substitutions, wherein the substitutions are made atpositions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8. In somepreferred embodiments, the cleaning compositions comprise at least onevariant serine protease, wherein the serine protease has immunologicalcross-reactivity with the serine protease set forth in SEQ ID NO:8. Insome preferred embodiments, the substitutions are made at positionsequivalent to positions 1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,18, 19, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 54, 55, 56, 57, 59, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 99, 100, 101,103, 104, 105, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 121, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135,136, 137, 140, 141, 142, 143, 144, 145, 146, 147, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 170, 171, 172, 175, 176, 177, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, and 189, in a Cellulomonas 69B4 protease comprising theamino acid sequence set forth in SEQ ID NO:8. In some preferredembodiments, the cleaning compositions further comprise one or moreadditional enzymes or enzyme derivatives selected from the groupconsisting of proteases, amylases, lipases, mannanases, pectinases,cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides compositions comprising the serineprotease variants having amino acid sequences comprising at least twoamino acid substitutions, wherein the substitutions are made atpositions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8, and atleast one stabilizing agent. In some preferred embodiments, thecompositions are cleaning compositions. In some particularly preferredembodiments, the stabilizing agent is selected from borax, glycerol, andcompetitive inhibitors. In some further particularly preferredembodiments, the competitive inhibitors stabilize the serine proteasevariants to anionic surfactants. In some still further embodiments, theserine protease variant is an autolytically stable variant.

The present invention also provides cleaning compositions comprising atleast 0.0001 weight percent of the serine protease variants having aminoacid sequences comprising at least two amino acid substitutions, whereinthe substitutions are made at positions equivalent to the positions in aCellulomonas 69B4 protease comprising the amino acid sequence set forthin SEQ ID NO:8, and optionally, an adjunct ingredient. In some preferredembodiments, the compositions comprise from about 0.001 to about 0.5weight percent of at least one serine protease variant. In somepreferred embodiments, the compositions comprise from about 0.01 toabout 0.1 weight percent of the serine protease. In some additionalpreferred embodiments, the cleaning compositions comprise an adjunctingredient. In some still further preferred embodiments, the cleaningcompositions comprise a sufficient amount of a pH modifier to providethe composition with a neat pH of from about 3 to about 5, wherein thecompositions are essentially free of materials that hydrolyze at a pH offrom about 3 to about 5. In some alternative preferred embodiments, thecleaning compositions comprise materials that hydrolyze comprise asurfactant material. In some particularly preferred embodiments, thesurfactant material comprises a sodium alkyl sulfate surfactant thatcomprises an ethylene oxide moiety.

The present invention further provides cleaning compositions comprisingthe serine protease variants having amino acid sequences comprising atleast two amino acid substitutions, wherein the substitutions are madeat positions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8, wherein thecleaning composition is a liquid. In some alternative embodiments, thecleaning composition is a powder, granular or tablet composition.

The present invention further provides cleaning compositions comprisingthe serine protease variants having amino acid sequences comprising atleast two amino acid substitutions, wherein the substitutions are madeat positions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8, wherein thecleaning compositions further comprise a hydrogen peroxide source. Insome preferred embodiments, the hydrogen peroxide source comprises atleast one persalt, wherein the persalt is alkalimetal perborate,alkalimetal percarbonate, alkalimetal perphosphate, alkalimetalpersulfate, or a mixture thereof. In some particularly preferredembodiments, the cleaning compositions further comprise a bleachcatalyst, bleach activator and/or mixtures thereof.

The present invention also provides methods of cleaning, comprising thesteps of: a) contacting a surface and/or an article comprising a fabricwith at least one cleaning composition of the present invention; and b)optionally washing and/or rinsing the surface or material.

The present invention also provides animal feeds comprising serineprotease variants having amino acid sequences comprising at least twoamino acid substitutions, wherein the substitutions are made atpositions equivalent to the positions in a Cellulomonas 69B4 proteasecomprising the amino acid sequence set forth in SEQ ID NO:8.

The present invention also provides textile and/or leather processingcompositions comprising serine protease variants having amino acidsequences comprising at least two amino acid substitutions, wherein thesubstitutions are made at positions equivalent to the positions in aCellulomonas 69B4 protease comprising the amino acid sequence set forthin SEQ ID NO:8.

The present invention also provides personal care compositionscomprising serine protease variants having amino acid sequencescomprising at least two amino acid substitutions, wherein thesubstitutions are made at positions equivalent to the positions in aCellulomonas 69B4 protease comprising the amino acid sequence set forthin SEQ ID NO:8.

DESCRIPTION OF THE FIGURES

FIG. 1 provides a map of the plasmid pHPLT-ASP-C1-2.

FIG. 2 provides a map of the plasmid pXX-KpnI.

FIG. 3 provides a map of the plasmid pHPLT.

FIG. 4 provides a map of the plasmid pUC18.

FIG. 5 provides a map of the plasmid pUC18-ASP

FIG. 6 provides a table showing the results from tergotometer testsperformed in the absence of HEPES Buffer (enzyme dosage was 0.55 ppm).

FIG. 7 provides a table showing the results from tergotometer testsperformed in the presence of HEPES buffer (enzyme dosage was 0.55 ppm).

DESCRIPTION OF THE INVENTION

The present invention provides novel Micrococcineae spp serine proteaseshaving multiple substitutions. In particular, the present inventionprovides serine proteases having multiple substitutions, DNA encodingthese proteases, vectors comprising the DNA encoding the proteases, hostcells transformed with the vector DNA, and enzymes produced by the hostcells. The present invention also provides cleaning compositions (e.g.,detergent compositions), animal feed compositions, and textile andleather processing compositions comprising these serine proteasevariants. In particularly preferred embodiments, the present inventionprovides mutant (i.e., variant) proteases derived from the wild-typeproteases described herein. These variant proteases also find use innumerous applications.

The present invention provides variant protease enzymes having multiplesubstitutions. Importantly, these variant enzymes have good stabilityand proteolytic activity. These variant enzymes find use in variousapplications, including but not limited to cleaning compositions, animalfeed, textile processing and etc. The present invention also providesmeans to produce these enzymes. In some preferred embodiments, thevariant proteases of the present invention are in pure or relativelypure form.

The present invention also provides nucleotide sequences which aresuitable to produce the variant proteases of the present invention inrecombinant organisms. In some embodiments, recombinant productionprovides means to produce the variant proteases in quantities that arecommercially viable.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, and recombinant DNA, which are within the skill of theart. Such techniques are known to those of skill in the art and aredescribed in numerous texts and reference works (See e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual”, Second Edition (ColdSpring Harbor), [1989]); and Ausubel et al., “Current Protocols inMolecular Biology” [1987]). All patents, patent applications, articlesand publications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For example,Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham,The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991)provide those of skill in the art with a general dictionaries of many ofthe terms used in the invention. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceof the present invention, the preferred methods and materials aredescribed herein. Accordingly, the terms defined immediately below aremore fully described by reference to the Specification as a whole. Also,as used herein, the singular “a”, “an” and “the” includes the pluralreference unless the context clearly indicates otherwise. Numeric rangesare inclusive of the numbers defining the range. Unless otherwiseindicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context theyare used by those of skill in the art.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of protein purification, molecularbiology, microbiology, recombinant DNA techniques and proteinsequencing, all of which are within the skill of those in the art.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.Additional definitions are provided in U.S. patent application Ser. No.10/576,331, which is incorporated by reference in its entirety.

I. Definitions

As used herein, the terms “protease,” and “proteolytic activity” referto a protein or peptide exhibiting the ability to hydrolyze peptides orsubstrates having peptide linkages. Many well known procedures exist formeasuring proteolytic activity (Kalisz, “Microbial Proteinases,” In:Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology,[1988]). For example, proteolytic activity may be ascertained bycomparative assays which analyze the respective protease's ability tohydrolyze a commercial substrate. Exemplary substrates useful in theanalysis of protease or protelytic activity, include, but are notlimited to di-methyl casein (Sigma C-9801), bovine collagen (SigmaC-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICNBiomedical 902111). Colorimetric assays utilizing these substrates arewell known in the art (See e.g., WO 99/34011; and U.S. Pat. No.6,376,450, both of which are incorporated herein by reference). The pNAassay (See e.g., Del Mar et al., Anal. Biochem., 99:316-320 [1979]) alsofinds use in determining the active enzyme concentration for fractionscollected during gradient elution. This assay measures the rate at whichp-nitroaniline is released as the enzyme hydrolyzes the solublesynthetic substrate,succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide(sAAPF-pNA). The rate of production of yellow color from the hydrolysisreaction is measured at 410 nm on a spectrophotometer and isproportional to the active enzyme concentration. In addition, absorbancemeasurements at 280 nm can be used to determine the total proteinconcentration. The active enzyme/total-protein ratio gives the enzymepurity.

As used herein, the terms “ASP protease,” “Asp protease,” and “Asp,”refer to the serine proteases described herein. In some preferredembodiments, the Asp protease is the protease designed herein as 69B4protease obtained from Cellulomonas strain 69B4. Thus, in preferredembodiments, the term “69B4 protease” refers to a naturally occurringmature protease derived from Cellulomonas strain 69B4 (DSM 16035) havingsubstantially identical amino acid sequences as provided in SEQ ID NO:8.In alternative embodiments, the present invention provides portions ofthe ASP protease.

The term “Cellulomonas protease homologues” refers to naturallyoccurring proteases having substantially identical amino acid sequencesto the mature protease derived from Cellulomonas strain 69B4 orpolynucleotide sequences which encode for such naturally occurringproteases, and which proteases retain the functional characteristics ofa serine protease encoded by such nucleic acids. In some embodiments,these protease homologues are referred to as “cellulomonadins.”

As used herein, the terms “protease variant,” “ASP variant,” “ASPprotease variant,” and “69B protease variant” are used in reference toproteases that are similar to the wild-type ASP, particularly in theirfunction, but have mutations (e.g., substitutions) in their amino acidsequence that make them different in sequence from the wild-typeprotease. Some of the amino acid residues identified for substitutionare conserved residues whereas others are not. In some embodiments, theprotease variants of the present invention include the mature forms ofprotease variants, while in other embodiments, the present inventionprovides the pro- and prepro-forms of such protease variants.

As used herein, “Cellulomonas ssp.” refers to all of the species withinthe genus “Cellulomonas,” which are Gram-positive bacteria classified asmembers of the Family Cellulomonadaceae, Suborder Micrococcineae, OrderActinomycetales, Class Actinobacteria. It is recognized that the genusCellulomonas continues to undergo taxonomical reorganization. Thus, itis intended that the genus include species that have been reclassified.

As used herein, “Streptomyces ssp.” refers to all of the species withinthe genus “Streptomyces,” which are Gram-positive bacteria classified asmembers of the Family Streptomycetaceae, Suborder Streptomycineae, OrderActinomycetales, class Actinobacteria. It is recognized that the genusStreptomyces continues to undergo taxonomical reorganization. Thus, itis intended that the genus include species that have been reclassified.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include, but arenot limited to, a single-, double- or triple-stranded DNA, genomic DNA,cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidinebases, or other natural, chemically, biochemically modified, non-naturalor derivatized nucleotide bases. The following are non-limiting examplesof polynucleotides: genes, gene fragments, chromosomal fragments, ESTs,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. In some embodiments, polynucleotides comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. In alternative embodiments, thesequence of nucleotides is interrupted by non-nucleotide components.

As used herein the term “gene” refers to a polynucleotide (e.g., a DNAsegment), that encodes a polypeptide and includes regions preceding andfollowing the coding regions as well as intervening sequences (introns)between individual coding segments (exons).

As used herein, “homologous genes” refers to a pair of genes fromdifferent, but usually related species, which correspond to each otherand which are identical or very similar to each other. The termencompasses genes that are separated by speciation (i.e., thedevelopment of new species) (e.g., orthologous genes), as well as genesthat have been separated by genetic duplication (e.g., paralogousgenes).

As used herein, “homology” refers to sequence similarity or identity,with identity being preferred. This homology is determined usingstandard techniques known in the art (See e.g., Smith and Waterman, Adv.Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443[1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988];programs such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al., Nucl. Acid Res., 12:387-395 [1984]).

As used herein, an “analogous sequence” is one wherein the function ofthe gene is essentially the same as the gene based on the Cellulomonasstrain 69B4 protease. Additionally, analogous genes include at least45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% sequence identity with the sequence of the Cellulomonas strain 69B4protease. Alternately, analogous sequences have an alignment of between70 to 100% of the genes found in the Cellulomonas strain 69B4 proteaseregion and/or have at least between 5-10 genes found in the regionaligned with the genes in the Cellulomonas strain 69B4 chromosome. Inadditional embodiments more than one of the above properties applies tothe sequence. Analogous sequences are determined by known methods ofsequence alignment. A commonly used alignment method is BLAST, althoughas indicated above and below, there are other methods that also find usein aligning sequences.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention. “Recombination,” “recombining,” and generating a“recombined” nucleic acid are generally the assembly of two or morenucleic acid fragments wherein the assembly gives rise to a chimericgene.

In a preferred embodiment, mutant DNA sequences are generated with sitesaturation mutagenesis in at least one codon. In another preferredembodiment, site saturation mutagenesis is performed for two or morecodons. In a further embodiment, mutant DNA sequences have more than50%, more than 55%, more than 60%, more than 65%, more than 70%, morethan 75%, more than 80%, more than 85%, more than 90%, more than 95%, ormore than 98% homology with the wild-type sequence. In alternativeembodiments, mutant DNA is generated in vivo using any known mutagenicprocedure such as, for example, radiation, nitrosoguanidine and thelike. The desired DNA sequence is then isolated and used in the methodsprovided herein.

As used herein “amino acid” refers to peptide or protein sequences orportions thereof.

As used herein, “protein of interest” and “polypeptide of interest”refer to a protein/polypeptide that is desired and/or being assessed. Insome embodiments, the protein of interest is expressed intracellularly,while in other embodiments, it is a secreted polypeptide. Inparticularly preferred embodiments, these enzyme include the serineproteases of the present invention. In some embodiments, the protein ofinterest is a secreted polypeptide which is fused to a signal peptide(i.e., an amino-terminal extension on a protein to be secreted). Nearlyall secreted proteins use an amino-terminal protein extension whichplays a crucial role in the targeting to and translocation of precursorproteins across the membrane. This extension is proteolytically removedby a signal peptidase during or immediately following membrane transfer.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in the host cell. Examples ofheterologous proteins include enzymes such as hydrolases includingproteases. In some embodiments, the gene encoding the proteins arenaturally occurring genes, while in other embodiments, mutated and/orsynthetic genes are used.

As used herein, “homologous protein” refers to a protein or polypeptidenative or naturally occurring in a cell. In preferred embodiments, thecell is a Gram-positive cell, while in particularly preferredembodiments, the cell is a Bacillus host cell. In alternativeembodiments, the homologous protein is a native protein produced byother organisms, including but not limited to E. coli, Streptomyces,Trichoderma, and Aspergillus. The invention encompasses host cellsproducing the homologous protein via recombinant DNA technology.

The terms “protein” and “polypeptide” are used interchangeabilityherein. The 3-letter code for amino acids as defined in conformity withthe IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) isused through out this disclosure. It is also understood that apolypeptide may be coded for by more than one nucleotide sequence due tothe degeneracy of the genetic code.

“Naturally occurring enzyme” refers to an enzyme having the unmodifiedamino acid sequence identical to that found in nature. Naturallyoccurring enzymes include native enzymes, those enzymes naturallyexpressed or found in the particular microorganism.

The terms “derived from” and “obtained from” refer to not only aprotease produced or producible by a strain of the organism in question,but also a protease encoded by a DNA sequence isolated from such strainand produced in a host organism containing such DNA sequence.Additionally, the term refers to a protease which is encoded by a DNAsequence of synthetic and/or cDNA origin and which has the identifyingcharacteristics of the protease in question. To exemplify, “proteasesderived from Cellulomonas” refers to those enzymes having proteolyticactivity which are naturally-produced by Cellulomonas, as well as toserine proteases like those produced by Cellulomonas sources but whichthrough the use of genetic engineering techniques are produced bynon-Cellulomonas organisms transformed with a nucleic acid encoding theserine proteases.

A “derivative” within the scope of this definition generally retains thecharacteristic proteolytic activity observed in the wild-type, native orparent form to the extent that the derivative is useful for similarpurposes as the wild-type, native or parent form. Functional derivativesof serine protease encompass naturally occurring, synthetically orrecombinantly produced peptides or peptide fragments which have thegeneral characteristics of the serine protease of the present invention.

The term “functional derivative” refers to a derivative of a nucleicacid which has the functional characteristics of a nucleic acid whichencodes serine protease. Functional derivatives of a nucleic acid whichencode serine protease of the present invention encompass naturallyoccurring, synthetically or recombinantly produced nucleic acids orfragments and encode serine protease characteristic of the presentinvention. Wild type nucleic acid encoding serine proteases according tothe invention include naturally occurring alleles and homologues basedon the degeneracy of the genetic code known in the art.

The term “identical” in the context of two nucleic acids or polypeptidesequences refers to the residues in the two sequences that are the samewhen aligned for maximum correspondence, as measured using one of thefollowing sequence comparison or analysis algorithms.

The term “optimal alignment” refers to the alignment giving the highestpercent identity score.

“Percent sequence identity,” “percent amino acid sequence identity,”“percent gene sequence identity,” and/or “percent nucleicacid/polynucleotide sequence identity,” with respect to two amino acid,polynucleotide and/or gene sequences (as appropriate), refer to thepercentage of residues that are identical in the two sequences when thesequences are optimally aligned. Thus, 80% amino acid sequence identitymeans that 80% of the amino acids in two optimally aligned polypeptidesequences are identical.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides thus refers to a polynucleotide or polypeptide thatcomprising at least 70% sequence identity, preferably at least 75%,preferably at least 80%, preferably at least 85%, preferably at least90%, preferably at least 95%, preferably at least 97%, preferably atleast 98% and preferably at least 99% sequence identity as compared to areference sequence using the programs or algorithms (e.g., BLAST, ALIGN,CLUSTAL) using standard parameters. One indication that two polypeptidesare substantially identical is that the first polypeptide isimmunologically cross-reactive with the second polypeptide. Typically,polypeptides that differ by conservative amino acid substitutions areimmunologically cross-reactive. Thus, a polypeptide is substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by a conservative substitution. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules hybridize to each other under stringent conditions (e.g.,within a range of medium to high stringency).

The phrase “equivalent,” in this context, refers to serine proteasesenzymes that are encoded by a polynucleotide capable of hybridizing tothe polynucleotide having the sequence as shown in SEQ ID NO:1, underconditions of medium to maximal stringency. For example, beingequivalent means that an equivalent mature serine protease comprises atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% and/or at least 99% sequenceidentity to the mature Cellulomonas serine protease having the aminoacid sequence of SEQ ID NO:8.

The term “isolated” or “purified” refers to a material that is removedfrom its original environment (e.g., the natural environment, if it isnaturally occurring). For example, the material is said to be “purified”when it is present in a particular composition in a higher concentrationthan exists in a naturally occurring or wild type organism or incombination with components not normally present upon expression from anaturally occurring or wild type organism. For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. Such polynucleotides could be part of a vector,and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. In preferred embodiments, a nucleicacid or protein is said to be purified, for example, if it gives rise toessentially one band in an electrophoretic gel or blot.

The term “isolated,” when used in reference to a DNA sequence, refers toa DNA sequence that has been removed from its natural genetic milieu andis thus free of other extraneous or unwanted coding sequences, and is ina form suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (See e.g., Dynan and Tijan, Nature 316:774-78 [1985]).The term “an isolated DNA sequence” is alternatively referred to as “acloned DNA sequence”.

The term “isolated,” when used in reference to a protein, refers to aprotein that is found in a condition other than its native environment.In a preferred form, the isolated protein is substantially free of otherproteins, particularly other homologous proteins. An isolated protein ismore than 10% pure, preferably more than 20% pure, and even morepreferably more than 30% pure, as determined by SDS-PAGE. Furtheraspects of the invention encompass the protein in a highly purified form(i.e., more than 40% pure, more than 60% pure, more than 80% pure, morethan 90% pure, more than 95% pure, more than 97% pure, and even morethan 99% pure), as determined by SDS-PAGE.

As used herein, the term “combinatorial mutagenesis” refers to methodsin which libraries of variants of a starting sequence are generated. Inthese libraries, the variants contain one or several mutations (e.g.,substitutions) chosen from a predefined set of mutations. In addition,the methods provide means to introduce random mutations which were notmembers of the predefined set of mutations (e.g., substitutions). Insome embodiments, the methods include those set forth in U.S. patentapplication Ser. No. 09/699,250, filed Oct. 26, 2000, herebyincorporated by reference. In alternative embodiments, combinatorialmutagenesis methods encompass commercially available kits (e.g.,QuikChange® Multisite, Stratagene, San Diego, Calif.).

As used herein, the term “library of mutants” refers to a population ofcells which are identical in most of their genome but include differenthomologues of one or more genes. Such libraries can be used, forexample, to identify genes or operons with improved traits.

As used herein, the term “starting gene” refers to a gene of interestthat encodes a protein of interest that is to be improved and/or changedusing the present invention.

As used herein, the term “multiple sequence alignment” (“MSA”) refers tothe sequences of multiple homologs of a starting gene that are alignedusing an algorithm (e.g., Clustal W).

As used herein, the terms “consensus sequence” and “canonical sequence”refer to an archetypical amino acid sequence against which all variantsof a particular protein or sequence of interest are compared. The termsalso refer to a sequence that sets forth the nucleotides that are mostoften present in a DNA sequence of interest. For each position of agene, the consensus sequence gives the amino acid that is most abundantin that position in the MSA.

As used herein, the term “consensus mutation” refers to a difference inthe sequence of a starting gene and a consensus sequence. Consensusmutations are identified by comparing the sequences of the starting geneand the consensus sequence resulting from an MSA. In some embodiments,consensus mutations are introduced into the starting gene such that itbecomes more similar to the consensus sequence. Consensus mutations alsoinclude amino acid changes that change an amino acid in a starting geneto an amino acid that is more frequently found in an MSA at thatposition relative to the frequency of that amino acid in the startinggene. Thus, the term consensus mutation comprises all single amino acidchanges that replace an amino acid of the starting gene with an aminoacid that is more abundant than the amino acid in the MSA.

As used herein, the term “initial hit” refers to a variant that wasidentified by screening a combinatorial consensus mutagenesis library.In preferred embodiments, initial hits have improved performancecharacteristics, as compared to the starting gene.

As used herein, the term “improved hit” refers to a variant that wasidentified by screening an enhanced combinatorial consensus mutagenesislibrary.

As used herein, the terms “improving mutation” and“performance-enhancing mutation” (“improving substitution” and“performance-enhancing substitution”) refer to a mutation (e.g.,substitution) that leads to improved performance when it is introducedinto the starting gene. In some preferred embodiments, these mutations(e.g., substitutions) are identified by sequencing hits that wereidentified during the screening step of the method. In most embodiments,mutations (e.g., substitutions) that are more frequently found in hitsare likely to be improving mutations (e.g., substitutions) as comparedto an unscreened combinatorial consensus mutagenesis library.

As used herein, the term “enhanced combinatorial consensus mutagenesislibrary” refers to a CCM library that is designed and constructed basedon screening and/or sequencing results from an earlier round of CCMmutagenesis and screening. In some embodiments, the enhanced CCM libraryis based on the sequence of an initial hit resulting from an earlierround of CCM. In additional embodiments, the enhanced CCM is designedsuch that mutations that were frequently observed in initial hits fromearlier rounds of mutagenesis and screening are favored. In somepreferred embodiments, this is accomplished by omitting primers thatencode performance-reducing mutations or by increasing the concentrationof primers that encode performance-enhancing mutations relative to otherprimers that were used in earlier CCM libraries.

As used herein, the term “performance-reducing mutations” refer tomutations in the combinatorial consensus mutagenesis library that areless frequently found in hits resulting from screening as compared to anunscreened combinatorial consensus mutagenesis library. In preferredembodiments, the screening process removes and/or reduces the abundanceof variants that contain “performance-reducing mutations.”

As used herein, the term “functional assay” refers to an assay thatprovides an indication of a protein's activity. In particularlypreferred embodiments, the term refers to assay systems in which aprotein is analyzed for its ability to function in its usual capacity.For example, in the case of enzymes, a functional assay involvesdetermining the effectiveness of the enzyme in catalyzing a reaction.

As used herein, the term “target property” refers to the property of thestarting gene that is to be altered. It is not intended that the presentinvention be limited to any particular target property. However, in somepreferred embodiments, the target property is the stability of a geneproduct (e.g., resistance to denaturation, proteolysis or otherdegradative factors), while in other embodiments, the level ofproduction in a production host is altered. Indeed, it is contemplatedthat any property of a starting gene will find use in the presentinvention.

The term “property” or grammatical equivalents thereof in the context ofa nucleic acid, as used herein, refer to any characteristic or attributeof a nucleic acid that can be selected or detected. These propertiesinclude, but are not limited to, a property affecting binding to apolypeptide, a property conferred on a cell comprising a particularnucleic acid, a property affecting gene transcription (e.g., promoterstrength, promoter recognition, promoter regulation, enhancer function),a property affecting RNA processing (e.g., RNA splicing, RNA stability,RNA conformation, and post-transcriptional modification), a propertyaffecting translation (e.g., level, regulation, binding of mRNA toribosomal proteins, post-translational modification). For example, abinding site for a transcription factor, polymerase, regulatory factor,etc., of a nucleic acid may be altered to produce desiredcharacteristics or to identify undesirable characteristics.

The term “property” or grammatical equivalents thereof in the context ofa polypeptide, as used herein, refer to any characteristic or attributeof a polypeptide that can be selected or detected. These propertiesinclude, but are not limited to oxidative stability, substratespecificity, catalytic activity, thermal stability, alkaline stability,pH activity profile, resistance to proteolytic degradation, K_(M),k_(cat), k_(cat)/k_(M) ratio, protein folding, inducing an immuneresponse, ability to bind to a ligand, ability to bind to a receptor,ability to be secreted, ability to be displayed on the surface of acell, ability to oligomerize, ability to signal, ability to stimulatecell proliferation, ability to inhibit cell proliferation, ability toinduce apoptosis, ability to be modified by phosphorylation orglycosylation, ability to treat disease.

As used herein, the term “screening” has its usual meaning in the artand is, in general a multi-step process. In the first step, a mutantnucleic acid or variant polypeptide therefrom is provided. In the secondstep, a property of the mutant nucleic acid or variant polypeptide isdetermined. In the third step, the determined property is compared to aproperty of the corresponding precursor nucleic acid, to the property ofthe corresponding naturally occurring polypeptide or to the property ofthe starting material (e.g., the initial sequence) for the generation ofthe mutant nucleic acid.

It will be apparent to the skilled artisan that the screening procedurefor obtaining a nucleic acid or protein with an altered property dependsupon the property of the starting material the modification of which thegeneration of the mutant nucleic acid is intended to facilitate. Theskilled artisan will therefore appreciate that the invention is notlimited to any specific property to be screened for and that thefollowing description of properties lists illustrative examples only.Methods for screening for any particular property are generallydescribed in the art. For example, one can measure binding, pH,specificity, etc., before and after mutation, wherein a change indicatesan alteration. Preferably, the screens are performed in ahigh-throughput manner, including multiple samples being screenedsimultaneously, including, but not limited to assays utilizing chips,phage display, and multiple substrates and/or indicators.

As used herein, in some embodiments, screens encompass selection stepsin which variants of interest are enriched from a population ofvariants. Examples of these embodiments include the selection ofvariants that confer a growth advantage to the host organism, as well asphage display or any other method of display, where variants can becaptured from a population of variants based on their binding orcatalytic properties. In a preferred embodiment, a library of variantsis exposed to stress (e.g., heat, protease, denaturation, etc.) andsubsequently variants that are still intact are identified in a screenor enriched by selection. It is intended that the term encompass anysuitable means for selection. Indeed, it is not intended that thepresent invention be limited to any particular method of screening.

As used herein, the term “targeted randomization” refers to a processthat produces a plurality of sequences where one or several positionshave been randomized. In some embodiments, randomization is complete(i.e., all four nucleotides, A, T, G, and C can occur at a randomizedposition). In alternative embodiments, randomization of a nucleotide islimited to a subset of the four nucleotides. Targeted randomization canbe applied to one or several codons of a sequence, coding for one orseveral proteins of interest. When expressed, the resulting librariesproduce protein populations in which one or more amino acid positionscan contain a mixture of all 20 amino acids or a subset of amino acids,as determined by the randomization scheme of the randomized codon. Insome embodiments, the individual members of a population resulting fromtargeted randomization differ in the number of amino acids, due totargeted or random insertion or deletion of codons. In furtherembodiments, synthetic amino acids are included in the proteinpopulations produced. In some preferred embodiments, the majority ofmembers of a population resulting from targeted randomization showgreater sequence homology to the consensus sequence than the startinggene. In some embodiments, the sequence encodes one or more proteins ofinterest. In alternative embodiments, the proteins have differingbiological functions. In some preferred embodiments, the incomingsequence comprises at least one selectable marker.

The terms “modified sequence” and “modified genes” are usedinterchangeably herein to refer to a sequence that includes a deletion,insertion or interruption of naturally occurring nucleic acid sequence.In some preferred embodiments, the expression product of the modifiedsequence is a truncated protein (e.g., if the modification is a deletionor interruption of the sequence). In some particularly preferredembodiments, the truncated protein retains biological activity. Inalternative embodiments, the expression product of the modified sequenceis an elongated protein (e.g., modifications comprising an insertioninto the nucleic acid sequence). In some embodiments, an insertion leadsto a truncated protein (e.g., when the insertion results in theformation of a stop codon). Thus, an insertion may result in either atruncated protein or an elongated protein as an expression product.

As used herein, the term “substutition” encompasses the replacement ofone amino acid in an amino acid sequence with another amino acid at thatposition in the amino acid sequence. Thus, the term encompassessituations in which insertion and/or deletion of another amino acid inan amino acid sequence changes the relative positions of the amino acidsin the sequence. Thus, while an amino acid may not be specificallytargeted for substitution (e.g., using the methods described herein),that amino acid may be “substituted” by another amino acid in thesequence due to a change in its relative position in the sequence. Forexample, by inserting an amino acid between the amino acids at positions1 and 2 of SEQ ID NO:8, the resulting sequence will be shifted by oneamino acid (i.e., the amino acid originally in position 2 is now inposition 3, etc.). It is intended that the term encompass multiple, aswell as single changes in the amino acid sequence (i.e., single ormultiple substitutions). It is also noted that as used herein, aminoacid substitutions are indicated by the naturally occurring amino acid,followed by the position, followed by the substituted amino acid. Thus,N024E (also indicated as N24E) indicates that the asparagine at position24 of SEQ ID NO:8 has been substituted with glutamic acid. As indicatedherein, multiple substitutions are indicated by either “/” or “−”. Thus,“R127A-G65Q” and “R127A/G65Q” both indicate that the amino acids atpositions 127 and 65 have been substituted (i.e., the arginine atposition 127 has been substituted with an alanine and the glycine atposition 65 has been substituted with a glutamine).

As used herein, the terms “mutant sequence” and “mutant gene” are usedinterchangeably and refer to a sequence that has an alteration in atleast one codon occurring in a host cell's wild-type sequence. Theexpression product of the mutant sequence is a protein with an alteredamino acid sequence relative to the wild-type. The expression productmay have an altered functional capacity (e.g., enhanced enzymaticactivity).

As used herein, the term “up mutation” refers to mutations for which ΔΔG values for a property are better than the parent protein (ΔΔ G values,<0).

As used herein, the term “down mutation” refers to mutations for whichΔΔ G values for a property are worse than the parent protein (ΔΔ Gvalues, >0).

As used herein, the term “productive site” refers to positions whichhave at least one up mutation for a given property.

As used herein, the term “unproductive site” refers to positions whichhave no up mutations for a given property.

The terms “mutagenic primer” or “mutagenic oligonucleotide” (usedinterchangeably herein) are intended to refer to oligonucleotidecompositions which correspond to a portion of the template sequence andwhich are capable of hybridizing thereto. With respect to mutagenicprimers, the primer will not precisely match the template nucleic acid,the mismatch or mismatches in the primer being used to introduce thedesired mutation into the nucleic acid library. As used herein,“non-mutagenic primer” or “non-mutagenic oligonucleotide” refers tooligonucleotide compositions which will match precisely to the templatenucleic acid. In one embodiment of the invention, only mutagenic primersare used. In another preferred embodiment of the invention, the primersare designed so that for at least one region at which a mutagenic primerhas been included, there is also non-mutagenic primer included in theoligonucleotide mixture. By adding a mixture of mutagenic primers andnon-mutagenic primers corresponding to at least one of the mutagenicprimers, it is possible to produce a resulting nucleic acid library inwhich a variety of combinatorial mutational patterns are presented. Forexample, if it is desired that some of the members of the mutant nucleicacid library retain their precursor sequence at certain positions whileother members are mutant at such sites, the non-mutagenic primersprovide the ability to obtain a specific level of non-mutant memberswithin the nucleic acid library for a given residue. The methods of theinvention employ mutagenic and non-mutagenic oligonucleotides which aregenerally between 10-50 bases in length, more preferably about 15-45bases in length. However, it may be necessary to use primers that areeither shorter than 10 bases or longer than 50 bases to obtain themutagenesis result desired. With respect to corresponding mutagenic andnon-mutagenic primers, it is not necessary that the correspondingoligonucleotides be of identical length, but only that there is overlapin the region corresponding to the mutation to be added.

Primers may be added in a pre-defined ratio according to the presentinvention. For example, if it is desired that the resulting library havea significant level of a certain specific mutation and a lesser amountof a different mutation at the same or different site, by adjusting theamount of primer added, it is possible to produce the desired biasedlibrary. Alternatively, by adding lesser or greater amounts ofnon-mutagenic primers, it is possible to adjust the frequency with whichthe corresponding mutation(s) are produced in the mutant nucleic acidlibrary.

As used herein, the phrase “contiguous mutations” refers to mutationswhich are presented within the same oligonucleotide primer. For example,contiguous mutations may be adjacent or nearby each other, however, theywill be introduced into the resulting mutant template nucleic acids bythe same primer.

As used herein, the phrase “discontiguous mutations” refers to mutationswhich are presented in separate oligonucleotide primers. For example,discontiguous mutations will be introduced into the resulting mutanttemplate nucleic acids by separately prepared oligonucleotide primers.

The terms “wild-type sequence,” or “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest that is the starting point ofa protein engineering project. The wild-type sequence may encode eithera homologous or heterologous protein. A homologous protein is one thehost cell would produce without intervention. A heterologous protein isone that the host cell would not produce but for the intervention.

As used herein, the term “antibodies” refers to immunoglobulins.Antibodies include but are not limited to immunoglobulins obtaineddirectly from any species from which it is desirable to produceantibodies. In addition, the present invention encompasses modifiedantibodies. The term also refers to antibody fragments that retain theability to bind to the epitope that the intact antibody binds andinclude polyclonal antibodies, monoclonal antibodies, chimericantibodies, anti-idiotype (anti-ID) antibodies. Antibody fragmentsinclude, but are not limited to the complementarity-determining regions(CDRs), single-chain fragment variable regions (scFv), heavy chainvariable region (VH), light chain variable region (VL). Polyclonal andmonoclonal antibodies are also encompassed by the present invention.Preferably, the antibodies are monoclonal antibodies.

The term “oxidation stable” refers to proteases of the present inventionthat retain a specified amount of enzymatic activity over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposed toor contacted with bleaching agents or oxidizing agents. In someembodiments, the proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after contactwith a bleaching or oxidizing agent over a given time period, forexample, at least 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes,16 minutes, 20 minutes, etc. In some embodiments, the stability ismeasured as described in the Examples.

The term “chelator stable” refers to proteases of the present inventionthat retain a specified amount of enzymatic activity over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposed toor contacted with chelating agents. In some embodiments, the proteasesretain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%,98% or 99% proteolytic activity after contact with a chelating agentover a given time period, for example, at least 10 minutes, 20 minutes,40 minutes, 60 minutes, 100 minutes, etc. In some embodiments, thechelator stability is measured as described in the Examples.

The terms “thermally stable” and “thermostable” refer to proteases ofthe present invention that retain a specified amount of enzymaticactivity after exposure to identified temperatures over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposedaltered temperatures. Altered temperatures includes increased ordecreased temperatures. In some embodiments, the proteases retain atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%proteolytic activity after exposure to altered temperatures over a giventime period, for example, at least 60 minutes, 120 minutes, 180 minutes,240 minutes, 300 minutes, etc. In some embodiments, the thermostabilityis determined as described in the Examples.

The term “enhanced stability” in the context of an oxidation, chelator,thermal and/or pH stable protease refers to a higher retainedproteolytic activity over time as compared to other serine proteases(e.g., subtilisin proteases) and/or wild-type enzymes.

The term “diminished stability” in the context of an oxidation,chelator, thermal and/or pH stable protease refers to a lower retainedproteolytic activity over time as compared to other serine proteases(e.g., subtilisin proteases) and/or wild-type enzymes.

The term “cleaning activity” refers to the cleaning performance achievedby the protease under conditions prevailing during the proteolytic,hydrolyzing, cleaning or other process of the invention. In someembodiments, cleaning performance is determined by the application ofvarious cleaning assays concerning enzyme sensitive stains, for examplegrass, blood, milk, or egg protein as determined by variouschromatographic, spectrophotometric or other quantitative methodologiesafter subjection of the stains to standard wash conditions. Exemplaryassays include, but are not limited to those described in WO 99/34011,and U.S. Pat. No. 6,605,458 (both of which are herein incorporated byreference), as well as those methods included in the Examples.

The term “cleaning effective amount” of a protease refers to thequantity of protease described hereinbefore that achieves a desiredlevel of enzymatic activity in a specific cleaning composition. Sucheffective amounts are readily ascertained by one of ordinary skill inthe art and are based on many factors, such as the particular proteaseused, the cleaning application, the specific composition of the cleaningcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, etc.

The term “cleaning adjunct materials,” as used herein, means any liquid,solid or gaseous material selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, granule,powder, bar, paste, spray, tablet, gel; or foam composition), whichmaterials are also preferably compatible with the protease enzyme usedin the composition. In some embodiments, granular compositions are in“compact” form, while in other embodiments, the liquid compositions arein a “concentrated” form.

The term “enhanced performance” in the context of cleaning activityrefers to an increased or greater cleaning activity of certain enzymesensitive stains such as egg, milk, grass or blood, as determined byusual evaluation after a standard wash cycle and/or multiple washcycles.

The term “diminished performance” in the context of cleaning activityrefers to an decreased or lesser cleaning activity of certain enzymesensitive stains such as egg, milk, grass or blood, as determined byusual evaluation after a standard wash cycle.

The term “comparative performance” in the context of cleaning activityrefers to at least 60%, at least 70%, at least 80% at least 90% at least95% of the cleaning activity of a comparative subtilisin protease (e.g.,commercially available proteases), including but not limited toOPTIMASE™ protease (Genencor), PURAFECT™ protease products (Genencor),SAVINASE™ protease (Novozymes), BPN′-variants (See e.g., U.S. Pat. No.Re 34,606), RELASE™, DURAZYME™, EVERLASE™, KANNASE™ protease(Novozymes), MAXACAL™, MAXAPEM™, PROPERASE™ proteases (Genencor; Seealso, U.S. Pat. No. Re 34,606, and U.S. Pat. Nos. 5,700,676; 5,955,340;6,312,936; and 6,482,628), and B. lentus variant protease products(e.g., those described in WO 92/21760, WO 95/23221 and/or WO 97/07770).Exemplary subtilisin protease variants include, but are not limited tothose having substitutions or deletions at residue positions equivalentto positions 76, 101, 103, 104, 120, 159, 167, 170, 194, 195, 217, 232,235, 236, 245, 248, and/or 252 of BPN′. Cleaning performance can bedetermined by comparing the proteases of the present invention withthose subtilisin proteases in various cleaning assays concerning enzymesensitive stains such as grass, blood or milk as determined by usualspectrophotometric or analytical methodologies after standard wash cycleconditions.

As used herein, a “low detergent concentration” system includesdetergents where less than about 800 ppm of detergent components arepresent in the wash water. Japanese detergents are typically consideredlow detergent concentration systems, as they have usually haveapproximately 667 ppm of detergent components present in the wash water.

As used herein, a “medium detergent concentration” systems includesdetergents wherein between about 800 ppm and about 2000 ppm of detergentcomponents are present in the wash water. North American detergents aregenerally considered to be medium detergent concentration systems asthey have usually approximately 975 ppm of detergent components presentin the wash water. Brazilian detergents typically have approximately1500 ppm of detergent components present in the wash water.

As used herein, “high detergent concentration” systems includesdetergents wherein greater than about 2000 ppm of detergent componentsare present in the wash water. European detergents are generallyconsidered to be high detergent concentration systems as they haveapproximately 3000-8000 ppm of detergent components in the wash water.

As used herein, “fabric cleaning compositions” include hand and machinelaundry detergent compositions including laundry additive compositionsand compositions suitable for use in the soaking and/or pretreatment ofstained fabrics (e.g., clothes, linens, and other textile materials).

As used herein, “non-fabric cleaning compositions” include non-textile(i.e., fabric) surface cleaning compositions, including but not limitedto dishwashing detergent compositions, oral cleaning compositions,denture cleaning compositions, and personal cleansing compositions.

The “compact” form of the cleaning compositions herein is best reflectedby density and, in terms of composition, by the amount of inorganicfiller salt. Inorganic filler salts are conventional ingredients ofdetergent compositions in powder form. In conventional detergentcompositions, the filler salts are present in substantial amounts,typically 17-35% by weight of the total composition. In contrast, incompact compositions, the filler salt is present in amounts notexceeding 15% of the total composition. In some embodiments, the fillersalt is present in amounts that do not exceed 10%, or more preferably,5%, by weight of the composition. In some embodiments, the inorganicfiller salts are selected from the alkali and alkaline-earth-metal saltsof sulfates and chlorides. A preferred filler salt is sodium sulfate.

II. Serine Protease Enzymes of the Present Invention and SequencesTherefor

The present invention provides isolated polynucleotides encoding aminoacid sequences which encode variant proteases. U.S. patent applicationSer. No. 10/576,331, incorporated by reference in its entirety providesvarious proteases, including the wild-type Cellulomonas serine protease,as well as numerous variant proteases, signal peptide coding sequences,and amino acid sequences. In some preferred embodiments of the presentinvention, the Cellulomonas spp. is Cellulomonas strain 69B4 (DSM16035),as described in U.S. patent application Ser. No. 10/576,331.

A. Serine Proteases

Although there may be variations in the sequence of a naturallyoccurring enzyme within a given species of organism, enzymes of aspecific type produced by organisms of the same species generally aresubstantially identical with respect to substrate specificity and/orproteolytic activity levels under given conditions (e.g., temperature,pH, water hardness, oxidative conditions, chelating conditions, andconcentration), etc. Thus, for the purposes of the present invention, itis contemplated that other strains and species of Cellulomonas alsoproduce the Cellulomonas protease of the present invention and thusprovide useful sources for the proteases of the present invention.Indeed, as presented herein, it is contemplated that other members ofthe Micrococcineae will find use in the present invention.

In some embodiments, the proteolytic polypeptides of this invention arecharacterized physicochemically, while in other embodiments, they arecharacterized based on their functionally, while in further embodiments,they are characterized using both sets of properties. Physicochemicalcharacterization takes advantages of well-known techniques such as SDSelectrophoresis, gel filtration, amino acid composition, massspectrometry (e.g., MALDI-TOF-MS, LC-ES-MS/MS, etc.), and sedimentationto determine the molecular weight of proteins, isoelectric focusing todetermine the pI of proteins, amino acid sequencing to determine theamino acid sequences of protein, crystallography studies to determinethe tertiary structures of proteins, and antibody binding to determineantigenic epitopes present in proteins.

In some embodiments, functional characteristics are determined bytechniques well known to the practitioner in the protease field andinclude, but are not limited to, hydrolysis of various commercialsubstrates, such as di-methyl casein (“DMC”) and/or AAPF-pNA. Thispreferred technique for functional characterization is described ingreater detail in the Examples provided herein.

The mature protease also displays proteolytic activity (e.g., hydrolyticactivity on a substrate having peptide linkages) such as DMC. In furtherembodiments, proteases of the present invention provide enhanced washperformance under identified conditions. Although the present inventionencompasses the protease 69B as described herein, in some embodiments,the proteases of the present invention exhibit at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activityas compared to the proteolytic activity of 69B4. In some embodiments,the proteases display at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%,95%, 96%, 97%, 98% or 99% proteolytic activity as compared to theproteolytic activity of proteases sold under the tradenames SAVINASE®(Novzymes) or PURAFECT® (Genencor) under the same conditions. In someembodiments, the proteases of the present invention display comparativeor enhanced wash performance under identified conditions as compared to69B4 under the same conditions. In some preferred embodiments, theproteases of the present invention display comparative or enhanced washperformance under identified conditions, as compared to proteases soldunder the tradenames SAVINASE® (Novozymes) or PURAFECT® (Genencor) underthe same conditions.

In yet further embodiments, the proteases and/or polynucleotidesencoding the proteases of the present invention are provided in purifiedform (i.e., present in a particular composition in a higher or lowerconcentration than exists in a naturally occurring or wild typeorganism), or in combination with components not normally present uponexpression from a naturally occurring or wild-type organism. However, itis not intended that the present invention be limited to proteases ofany specific purity level, as ranges of protease purity find use invarious applications in which the proteases of the present invention aresuitable.

B. Nucleic and Amino Acid Sequences of Serine Proteases

The DNA sequence of the asp gene (SEQ ID NO:1) derived from Cellulomonasstrain 69B4 (DSM 16035) is provided below. The initiating polynucleotideencoding the signal peptide of the Cellulomonas strain 69B4 protease isin bold (ATG). This sequence also includes the signal peptide andprecursor serine protease.

The following DNA sequence (SEQ ID NO:2) encodes the signal peptide (SEQID NO:9) that is operatively linked to the precursor protease (SEQ IDNO:7) derived from Cellulomonas strain 69B4 (DSM 16035). The initiatingpolynucleotide encoding the signal peptide of the Cellulomonas strain69B4 protease is in bold (ATG). The termination codon (TGA) begins withresidue 1486. Residues 85, 595, and 1162, relate to the initial residuesof the N terminal prosequence, mature sequence and Carboxyl terminalprosequence, respectively, are bolded and underlined.

(SEQ ID NO: 2)ATGACACCAC GCACAGTCAC GCGGGCCCTG GCCGTGGCCA CCGCAGCCGC CACACTCCTG   60GCAGGCGGCA TGGCCGCCCA GGCCAACGAG CCCGCACCAC CCGGGAGCGC GAGCGCACCG  120CCACGCCTGG CCGAGAAGCT CGACCCCGAC CTCCTCGAGG CCATGGAGCG CGACCTGGGC  180CTCGACGCGG AGGAAGCCGC CGCCACCCTG GCGTTCCAGC ACGACGCAGC CGAGACCGGC  240GAGGCCCTCG CCGAAGAGCT CGACGAGGAC TTCGCCGGCA CCTGGGTCGA GGACGACGTC  300CTGTACGTCG CCACCACCGA CGAGGACGCC GTCGAGGAGG TCGAGGGCGA AGGCGCCACG  360GCCGTCACCG TCGAGCACTC CCTGGCCGAC CTCGAGGCCT GGAAGACCGT CCTCGACGCC  420GCCCTCGAGG GCCACGACGA CGTGCCCACC TGGTACGTCG ACGTCCCGAC CAACAGCGTC  480GTCGTCGCCG TCAAGGCCGG AGCCCAGGAC GTCGCCGCCG GCCTCGTCGA AGGTGCCGAC  540GTCCCGTCCG ACGCCGTGAC CTTCGTCGAG ACCGACGAGA CCCCGCGGAC CATGTTCGAC  600GTGATCGGCG GCAACGCCTA CACCATCGGG GGGCGCAGCC GCTGCTCGAT CGGGTTCGCG  660GTCAACGGCG GGTTCATCAC CGCCGGCCAC TGCGGCCGCA CCGGCGCCAC CACCGCCAAC  720CCCACCGGGA CCTTCGCCGG GTCCAGCTTC CCGGGCAACG ACTACGCGTT CGTCCGTACC  780GGGGCCGGCG TGAACCTGCT GGCCCAGGTC AACAACTACT CCGGTGGCCG CGTCCAGGTC  840GCCGGGCACA CCGCGGCCCC CGTCGGCTCG GCCGTGTGCC GGTCCGGGTC GACCACCGGG  900TGGCACTGCG GCACCATCAC TGCGCTCAAC TCCTCGGTCA CCTACCCCGA GGGCACCGTC  960CGCGGCCTGA TCCGCACCAC CGTCTGCGCC GAGCCCGGCG ACTCCGGTGG CTCGCTGCTC 1020GCCGGCAACC AGGCCCAGGG CGTCACGTCC GGCGGCTCCG GCAACTGCCG CACCGGTGGC 1080ACCACGTTCT TCCAGCCGGT CAACCCCATC CTCCAGGCGT ACGGCCTGAG GATGATCACC 1140ACGGACTCGG GCAGCAGCCC GGCCCCTGCA CCGACCTCCT GCACCGGCTA CGCCCGCACC 1200TTCACCGGGA CCCTCGCGGC CGGCCGGGCC GCCGCCCAGC CCAACGGGTC CTACGTGCAG 1260GTCAACCGGT CCGGGACCCA CAGCGTGTGC CTCAACGGGC CCTCCGGTGC GGACTTCGAC 1320CTCTACGTGC AGCGCTGGAA CGGCAGCTCC TGGGTGACCG TCGCCCAGAG CACCTCCCCC 1380GGCTCCAACG AGACCATCAC CTACCGCGGC AACGCCGGCT ACTACCGCTA CGTGGTCAAC 1440GCCGCGTCCG GCTCCGGTGC CTACACCATG GGGCTCACCC TCCCCTGA              1488

The following DNA sequence (SEQ ID NO:3) encodes the precursor proteasederived from Cellulomonas strain 69B4 (DSM 16035).

(SEQ ID NO: 3) 1AACGAGCCCG CACCACCCGG GAGCGCGAGC GCACCGCCAC GCCTGGCCGA GAAGCTCGACTTGCTCGGGC GTGGTGGGCC CTCGCGCTCG CGTGGCGGTG CGGACCGGCT CTTCGAGCTG 61CCCGACCTCC TCGAGGCCAT GGAGCGCGAC CTGGGCCTCG ACGCGGAGGA AGCCGCCGCCGGGCTGGAGG AGCTCCGGTA CCTCGCGCTG GACCCGGAGC TGCGCCTCCT TCGGCGGCGG 121ACCCTGGCGT TCCAGCACGA CGCAGCCGAG ACCGGCGAGG CCCTCGCCGA AGAGCTCGACTGGGACCGCA AGGTCGTGCT GCGTCGGCTC TGGCCGCTCC GGGAGCGGCT TCTCGAGCTG 181GAGGACTTCG CCGGCACCTG GGTCGAGGAC GACGTCCTGT ACGTCGCCAC CACCGACGAGCTCCTGAAGC GGCCGTGGAC CCAGCTCCTG CTGCAGGACA TGCAGCGGTG GTGGCTGCTC 241GACGCCGTCG AGGAGGTCGA GGGCGAAGGC GCCACGGCCG TCACCGTCGA GCACTCCCTGCTGCGGCAGC TCCTCCAGCT CCCGCTTCCG CGGTGCCGGC AGTGGCAGCT CGTGAGGGAC 301GCCGACCTCG AGGCCTGGAA GACCGTCCTC GACGCCGCCC TCGAGGGCCA CGACGACGTGCGGCTGGAGC TCCGGACCTT CTGGCAGGAG CTGCGGCGGG AGCTCCCGGT GCTGCTGCAC 361CCCACCTGGT ACGTCGACGT CCCGACCAAC AGCGTCGTCG TCGCCGTCAA GGCCGGAGCCGGGTGGACCA TGCAGCTGCA GGGCTGGTTG TCGCAGCAGC AGCGGCAGTT CCGGCCTCGG 421CAGGACGTCG CCGCCGGCCT CGTCGAAGGT GCCGACGTCC CGTCCGACGC CGTGACCTTCGTCCTGCAGC GGCGGCCGGA GCAGCTTCCA CGGCTGCAGG GCAGGCTGCG GCACTGGAAG 481GTCGAGACCG ACGAGACCCC GCGGACCATG TTCGACGTGA TCGGCGGCAA CGCCTACACCCAGCTCTGGC TGCTCTGGGG CGCCTGGTAC AAGCTGCACT AGCCGCCGTT GCGGATGTGG 541ATCGGGGGGC GCAGCCGCTG CTCGATCGGG TTCGCGGTCA ACGGCGGGTT CATCACCGCCTAGCCCCCCG CGTCGGCGAC GAGCTAGCCC AAGCGCCAGT TGCCGCCCAA GTAGTGGCGG 601GGCCACTGCG GCCGCACCGG CGCCACCACC GCCAACCCCA CCGGGACCTT CGCCGGGTCCCCGGTGACGC CGGCGTGGCC GCGGTGGTGG CGGTTGGGGT GGCCCTGGAA GCGGCCCAGG 661AGCTTCCCGG GCAACGACTA CGCGTTCGTC CGTACCGGGG CCGGCGTGAA CCTGCTGGCCTCGAAGGGCC CGTTGCTGAT GCGCAAGCAG GCATGGCCCC GGCCGCACTT GGACGACCGG 721CAGGTCAACA ACTACTCCGG TGGCCGCGTC CAGGTCGCCG GGCACACCGC GGCCCCCGTCGTCCAGTTGT TGATGAGGCC ACCGGCGCAG GTCCAGCGGC CCGTGTGGCG CCGGGGGCAG 781GGCTCGGCCG TGTGCCGGTC CGGGTCGACC ACCGGGTGGC ACTGCGGCAC CATCACTGCGCCGAGCCGGC ACACGGCCAG GCCCAGCTGG TGGCCCACCG TGACGCCGTG GTAGTGACGC 841CTCAACTCCT CGGTCACCTA CCCCGAGGGC ACCGTCCGCG GCCTGATCCG CACCACCGTCGAGTTGAGGA GCCAGTGGAT GGGGCTCCCG TGGCAGGCGC CGGACTAGGC GTGGTGGCAG 901TGCGCCGAGC CCGGCGACTC CGGTGGCTCG CTGCTCGCCG GCAACCAGGC CCAGGGCGTCACGCGGCTCG GGCCGCTGAG GCCACCGAGC GACGAGCGGC CGTTGGTCCG GGTCCCGCAG 961ACGTCCGGCG GCTCCGGCAA CTGCCGCACC GGTGGCACCA CGTTCTTCCA GCCGGTCAACTGCAGGCCGC CGAGGCCGTT GACGGCGTGG CCACCGTGGT GCAAGAAGGT CGGCCAGTTG 1021CCCATCCTCC AGGCGTACGG CCTGAGGATG ATCACCACGG ACTCGGGCAG CAGCCCGGCCGGGTAGGAGG TCCGCATGCC GGACTCCTAC TAGTGGTGCC TGAGCCCGTC GTCGGGCCGG 1081CCTGCACCGA CCTCCTGCAC CGGCTACGCC CGCACCTTCA CCGGGACCCT CGCGGCCGGCGGACGTGGCT GGAGGACGTG GCCGATGCGG GCGTGGAAGT GGCCCTGGGA GCGCCGGCCG 1141CGGGCCGCCG CCCAGCCCAA CGGGTCCTAC GTGCAGGTCA ACCGGTCCGG GACCCACAGCGCCCGGCGGC GGGTCGGGTT GCCCAGGATG CACGTCCAGT TGGCCAGGCC CTGGGTGTCG 1201GTGTGCCTCA ACGGGCCCTC CGGTGCGGAC TTCGACCTCT ACGTGCAGCG CTGGAACGGCCACACGGAGT TGCCCGGGAG GCCACGCCTG AAGCTGGAGA TGCACGTCGC GACCTTGCCG 1261AGCTCCTGGG TGACCGTCGC CCAGAGCACC TCCCCCGGCT CCAACGAGAC CATCACCTACTCGAGGACCC ACTGGCAGCG GGTCTCGTGG AGGGGGCCGA GGTTGCTCTG GTAGTGGATG 1321CGCGGCAACG CCGGCTACTA CCGCTACGTG GTCAACGCCG CGTCCGGCTC CGGTGCCTACGCGCCGTTGC GGCCGATGAT GGCGATGCAC CAGTTGCGGC GCAGGCCGAG GCCACGGATG 1381ACCATGGGGC TCACCCTCCC CTGA TGGTACCCCG AGTGGGAGGG GACT

The following DNA sequence (SEQ ID NO:4) encodes the mature proteasederived from Cellulomonas strain 69B4 (DSM 16035).

(SEQ ID NO: 4) 1 TTCGACGTGA TCGGCGGCAA CGCCTACACC ATCGGGGGGC GCAGCCGCTG CTCGATCGGGAAGCTGCACT AGCCGCCGTT GCGGATGTGG TAGCCCCCCG CGTCGGCGAC GAGCTAGCCC 61TTCGCGGTCA ACGGCGGGTT CATCACCGCC GGCCACTGCG GCCGCACCGG CGCCACCACCAAGCGCCAGT TGCCGCCCAA GTAGTGGCGG CCGGTGACGC CGGCGTGGCC GCGGTGGTGG 121GCCAACCCCA CCGGGACCTT CGCCGGGTCC AGCTTCCCGG GCAACGACTA CGCGTTCGTCCGGTTGGGGT GGCCCTGGAA GCGGCCCAGG TCGAAGGGCC CGTTGCTGAT GCGCAAGCAG 181CGTACCGGGG CCGGCGTGAA CCTGCTGGCC CAGGTCAACA ACTACTCCGG TGGCCGCGTCGCATGGCCCC GGCCGCACTT GGACGACCGG GTCCAGTTGT TGATGAGGCC ACCGGCGCAG 241CAGGTCGCCG GGCACACCGC GGCCCCCGTC GGCTCGGCCG TGTGCCGGTC CGGGTCGACCGTCCAGCGGC CCGTGTGGCG CCGGGGGCAG CCGAGCCGGC ACACGGCCAG GCCCAGCTGG 301ACCGGGTGGC ACTGCGGCAC CATCACTGCG CTCAACTCCT CGGTCACCTA CCCCGAGGGCTGGCCCACCG TGACGCCGTG GTAGTGACGC GAGTTGAGGA GCCAGTGGAT GGGGCTCCCG 361ACCGTCCGCG GCCTGATCCG CACCACCGTC TGCGCCGAGC CCGGCGACTC CGGTGGCTCGTGGCAGGCGC CGGACTAGGC GTGGTGGCAG ACGCGGCTCG GGCCGCTGAG GCCACCGAGC 421CTGCTCGCCG GCAACCAGGC CCAGGGCGTC ACGTCCGGCG GCTCCGGCAA CTGCCGCACCGACGAGCGGC CGTTGGTCCG GGTCCCGCAG TGCAGGCCGC CGAGGCCGTT GACGGCGTGG 481GGTGGCACCA CGTTCTTCCA GCCGGTCAAC CCCATCCTCC AGGCGTACGG CCTGAGGATGCCACCGTGGT GCAAGAAGGT CGGCCAGTTG GGGTAGGAGG TCCGCATGCC GGACTCCTAC 561ATCACCACGG ACTCGGGCAG CAGCCCG TAGTGGTGCC TGAGCCCGTC GTCGGGC

The following DNA sequence (SEQ ID NO:5) encodes the signal peptidederived from Cellulomonas strain 69B4 (DSM 16035)

(SEQ ID NO: 5) 1ATGACACCAC CACAGTCAC GCGGGCCCTG GCCGTGGCCA CCGCAGCCGC CACACTCCTGTACTGTGGTG CGTGTCAGTG CGCCCGGGAC CGGCACCGGT GGCGTCGGCG GTGTGAGGAC 61GCAGGCGGCA TGGCCGCCCA GGCC CGTCCGCCGT ACCGGCGGGT CCGG

The following sequence is the amino acid sequence (SEQ ID NO:6) of thesignal sequence and precursor protease derived from Cellulomonas strain69B4 (DSM 16035), including the signal sequence [segments 1a-c](residues 1-28 [−198 to −171]), an N-terminal prosequence [segments2a-r] (residues 29-198 [−170 to −1]), a mature protease [segments 3a-t](residues 199-387 [1-189]), and a C-terminal prosequence [segments 4a-1](residues 388-495 [190-398]) encoded by the DNA sequences set forth inSEQ ID NOS:1, 2, 3 and 4. The N-terminal sequence of the mature proteaseamino acid sequence is in bold.

(SEQ ID NO: 6)  1 MTPRTVTRAL AVATAAATLL AGGMAAQA NE PAPPGSASAP PRLAEKLDPD    la       lb         lc      2a 2b         2c  51 LLEAMERDLG LDAEEAAATL AFQHDAAETG EALAEELDED FAGTWVEDDV        2d         2e         2f        2g          2h 101 LYVATTDEDA VEEVEGEGAT AVTVEHSLAD LEAWKTVLDA ALEGHDDVPT        2i         2j         2k        2l          2m 151 WYVDVPTNSV VVAVKAGAQD VAAGLVEGAD VPSDAVTFVE TDETPRTM  FD        2n         2o         2p        2q          2r   3a  201 VIGGNAYTIG   GRSRCSIGFA VNGGFITAGH CGRTGATTAN PTGTFAGSSF        3b         3c         3d        3e          3f 251 PGNDYAFVRT GAGVNLLAQV NNYSGGRVQV AGHTAAPVGS AVCRSGSTTG        3g         3h         3i        3j          3k 301 WHCGTITALN SSVTYPEGTV RGLIRTTVCA EPGDSGGSLL AGNQAQGVTS        3l         3m         3n        3o          3p351 GGSGNCRTGG TTFFQPVNPI LQAYGLRMIT TDSGSSP APA PTSCTGYART        3q         3r         3s        3t       4a 4b 401 FTGTLAAGRA AAQPNGSYVQ VNRSGTHSVC LNGPSGADFD LYVQRWNGSS        4c         4d         4e         4f         4g 451 WVTVAQSTSP GSNETITYRG NAGYYRYVVN AASGSGAYTM GLTLP         4h         4i         4j        4k        4l 

The following sequence (SEQ ID NO:7) is the amino acid sequence of theprecursor protease derived from Cellulomonas strain 69B4 (DSM 16035)(SEQ ID NO:7).

(SEQ ID NO: 7) 1 NEPAPPGSAS APPRLAEKLD PDLLEAMERD.LGLDAEEAAA. TLAFQHDAAE51 TGEALAEELD EDFAGTWVED DVLYVATTDE DAVEEVEGEG ATAVTVEHSL 101ADLEAWKTVL DAALEGHDDV PTWYVDVPTN SVVVAVKAGA QDVAAGLVEG 151ADVPSDAVTF VETDETPRTM FDVIGGNAYT IGGRSRCSIG FAVNGGFITA 201GHCGRTGATT ANPTGTFAGS SFPGNDYAFV RTGAGVNLLA QVNNYSGGRV 251QVAGHTAAPV GSAVCRSGST TGWHCGTITA LNSSVTYPEG TVRGLIRTTV 301CAEPGDSGGS LLAGNQAQGV TSGGSGNCRT GGTTFFQPVN PILQAYGLRM 351ITTDSGSSPA PAPTSCTGYA RTFTGTLAAG RAAAQPNGSY VQVNRSGTHS 401VCLNGPSGAD FDLYVQRWNG SSWVTVAQST SPGSNETITY RGNAGYYRYV 451VNAASGSGAY TMGLTLP 

The following sequence (SEQ ID NO:8). is the amino acid sequence of themature protease derived from Cellulomonas strain 69B4 (DSM 16035). Thecatalytic triad residues H32, D56 and S132 are bolded and underlined.

(SEQ ID NO: 8) 1 FDVIGGNAYT IGGRSRCSIG FAVNGGFITA G H CGRTGATTANPTGTFAGS 51 SFPGN D YAFV RTGAGVNLLA QVNNYSGGRV QVAGHTAAPV GSAVCRSGST101 TGWHCGTITA LNSSVTYPEG TVRGLIRTTV CAEPGD S GGS LLAGNQAQGV 151TSGGSGNCRT GGTTFFQPVN PILQAYGLRM ITTDSGSSP

The following sequence (SEQ ID NO:9) is the amino acid sequence of thesignal peptide of the protease derived from Cellulomonas strain 69B4(DSM 16035).

(SEQ ID NO: 9) 1 MTPRTVTRAL AVATAAATLL AGGMAAQA

In some embodiments, the present invention encompasses variants thatcomprise at least a portion of the approximately 1621 base pairs inlength polynucleotide set forth in SEQ. ID NO:1. In some particularlypreferred embodiments, the present invention provides variants that havemultiple mutations as compared to the wild-type (e.g., “parent”) serineprotease. In some of these more particularly preferred embodiments,these multiply mutated variants exhibit improved performance, ascompared to the wild-type (e.g., “parent”) serine protease.

As will be understood by the skilled artisan, due to the degeneracy ofthe genetic code, a variety of polynucleotides can encode the signalpeptide, precursor protease and/or mature protease provided hereinand/or in U.S. patent application Ser. No. 10/576,331, incorporated byreference in its entirety (e.g., SEQ ID NOS:6, 7, and/or 8 of U.S.patent application Ser. No. 10/576,331) or a protease having the %sequence identity described above. Another embodiment of the presentinvention encompasses a polynucleotide comprising a nucleotide sequencehaving at least 70% sequence identity, at least 75% sequence identity,at least 80% sequence identity, at least 85% sequence identity, at least90% sequence identity, at least 92% sequence identity, at least 95%sequence identity, at least 97% sequence identity, at least 98% sequenceidentity and at least 99% sequence identity to the polynucleotidesequence of SEQ ID NOS:2, 3, and/or 4, respectively, encoding the signalpeptide and precursor protease, the precursor protease and/or the matureprotease, respectively.

In additional embodiments, the present invention provides fragments orportions of DNA that encodes proteases, so long as the encoded fragmentretains proteolytic activity. Another embodiment of the presentinvention encompasses polynucleotides having at least 20% of thesequence length, at least 30% of the sequence length, at least 40% ofthe sequence length, at least 50% of the sequence length, at least 60%of the sequence length, 70% of the sequence length, at least 75% of thesequence length, at least 80% of the sequence length, at least 85% ofthe sequence length, at least 90% of the sequence length, at least 92%of the sequence length, at least 95% of the sequence length, at least97% of the sequence length, at least 98% of the sequence length and atleast 99% of the sequence of the polynucleotide sequence of SEQ ID NO:1,encoding the precursor protease. In alternative embodiments, thesefragments or portions of the sequence length are contiguous portions ofthe sequence length, useful for shuffling of the DNA sequence inrecombinant DNA sequences (See e.g., U.S. Pat. No. 6,132,970)

Another embodiment of the invention includes fragments of the DNAdescribed herein that find use according to art recognized techniques inobtaining partial length DNA fragments capable of being used to isolateor identify polynucleotides encoding mature protease enzyme describedherein from Cellulomonas 69B4, or a segment thereof having proteolyticactivity. Moreover, the DNA provided in SEQ ID NO:1 finds use inidentifying homologous fragments of DNA from other species, andparticularly from Cellulomonas spp. which encode a protease or portionthereof having proteolytic activity.

In addition, the present invention encompasses using primer or probesequences constructed from SEQ ID NO:1, or a suitable portion orfragment thereof (e.g., at least about 5-20 or 10-15 contiguousnucleotides), as a probe or primer for screening nucleic acid of eithergenomic or cDNA origin. In some embodiments, the present inventionprovides DNA probes of the desired length (i.e., generally between 100and 1000 bases in length), based on the sequences in SEQ ID NO:1.

In some embodiments, the DNA fragments are electrophoretically isolated,cut from the gel, and recovered from the agar matrix of the gel. Inpreferred embodiments, this purified fragment of DNA is then labeled(using, for example, the Megaprime labeling system according to theinstructions of the manufacturer) to incorporate P³² in the DNA. Thelabeled probe is denatured by heating to 95° C. for a given period oftime (e.g., 5 minutes), and immediately added to the membrane andprehybridization solution. The hybridization reaction proceeds for anappropriate time and under appropriate conditions (e.g., 18 hours at 37°C.), with gentle shaking or rotation. The membrane is rinsed (e.g.,twice in SSC/0.3% SDS) and then washed in an appropriate wash solutionwith gentle agitation. The stringency desired is a reflection of theconditions under which the membrane (filter) is washed. In someembodiments herein, “low-stringency” conditions involve washing with asolution of 0.2×SSC/0.1% SDS at 20° C. for 15 minutes, while in otherembodiments, “medium-stringency” conditions, involve a further washingstep comprising washing with a solution of 0.2×SSC/0.1% SDS at 37° C.for 30 minutes, while in other embodiments, “high-stringency” conditionsinvolve a further washing step comprising washing with a solution of0.2×SSC/0.1% SDS at 37° C. for 45 minutes, and in further embodiments,“maximum-stringency” conditions involve a further washing stepcomprising washing with a solution of 0.2×SSC/0.1% SDS at 37° C. for 60minutes. Thus, various embodiments of the present invention providepolynucleotides capable of hybridizing to a probed derived from thenucleotide sequence provided in SEQ ID NOS:1 or 2, under conditions ofmedium, high and/or maximum stringency.

After washing, the membrane is dried and the bound probe detected. IfP³² or another radioisotope is used as the labeling agent, the boundprobe is detected by autoradiography. Other techniques for thevisualization of other probes are well-known to those of skill in theart. The detection of a bound probe indicates a nucleic acid sequencehas the desired homology, and therefore identity, to any sequence ofinterest provided herein is encompassed by the present invention.Accordingly, the present invention provides methods for the detection ofnucleic acid encoding a protease encompassed by the present inventionwhich comprises hybridizing part or all of a nucleic acid sequence ofSEQ ID NO:1 with other nucleic acid of either genomic or cDNA origin.

As indicated above, in other embodiments, hybridization conditions arebased on the melting temperature (Tm) of the nucleic acid bindingcomplex, to confer a defined “stringency” as explained below. “Maximumstringency” typically occurs at about Tm−5° C. (5° C. below the Tm ofthe probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. As known to those ofskill in the art, medium, high and/or maximum stringency hybridizationare chosen such that conditions are optimized to identify or detectpolynucleotide sequence homologues or equivalent polynucleotidesequences.

In yet additional embodiments, the present invention provides nucleicacid constructs (i.e., expression vectors) comprising thepolynucleotides encoding the proteases of the present invention. Infurther embodiments, the present invention provides host cellstransformed with at least one of these vectors.

In further embodiments, the present invention provides polynucleotidesequences further encoding a signal sequence, as described in U.S.patent application Ser. No. 10/576,331, incorporated by reference in itsentirety. In some of these embodiments, the present invention provides asequence with a putative signal sequence, and polynucleotides beingcapable of hybridizing to a probe derived from the nucleotide sequencedisclosed in SEQ ID NO:1 under conditions of medium, high and/or maximalstringency, wherein the signal sequences have substantially the samesignal activity as the signal sequence encoded by the polynucleotide ofthe present invention.

In some embodiments, the signal activity is indicated by substantiallythe same level of secretion of the protease into the fermentationmedium, as the starting material, as described in U.S. patentapplication Ser. No. 10/576,331. Additional means for determining thelevels of secretion of a heterologous or homologous protein in aGram-positive host cell and detecting secreted proteins include usingeither polyclonal or monoclonal antibodies specific for the protein.Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS), aswell-known to those in the art.

Further aspects of the present invention encompass polypeptides havingproteolytic activity comprising 65% amino acid sequence identity, atleast 70% sequence identity, at least 75% amino acid sequence identity,at least 80% amino acid sequence identity, at least 85% amino acidsequence identity, at least 90% amino acid sequence identity, at least92% amino acid sequence identity, at least 95% amino acid sequenceidentity, at least 97% amino acid sequence identity, at least 98% aminoacid sequence identity and at least 99% amino acid sequence identity tothe amino acid sequence of SEQ ID NOS:7 or 8, and as described hereinand in U.S. patent application Ser. No. 10/576,331. The proteolyticactivity of these polypeptides is determined using methods known in theart and include such methods as those used to assess detergent function.In further embodiments, the polypeptides are isolated.

III. Obtaining Polynucleotides Encoding Micrococcineae (E.g.,Cellulomonas) Proteases of the Present Invention

In some embodiments, nucleic acid encoding a protease of the presentinvention is obtained by standard procedures known in the art from, forexample, cloned DNA (e.g., a DNA “library”), chemical synthesis, cDNAcloning, PCR, cloning of genomic DNA or fragments thereof, or purifiedfrom a desired cell, such as a bacterial or fungal species (See, forexample, Sambrook et al., supra [1989]; and Glover and Hames (eds.), DNACloning: A Practical Approach, Vols. 1 and 2, Second Edition). Synthesisof polynucleotide sequences is well known in the art (See e.g., Beaucageand Caruthers, Tetrahedron Lett., 22:1859-1862 [1981]), including theuse of automated synthesizers (See e.g., Needham-VanDevanter et al.,Nucl. Acids Res., 12:6159-6168 [1984]). DNA sequences can also be custommade and ordered from a variety of commercial sources. As described ingreater detail herein, in some embodiments, nucleic acid sequencesderived from genomic DNA contain regulatory regions in addition tocoding regions.

In some embodiments involving the molecular cloning of the gene fromgenomic DNA, DNA fragments are generated, some of which comprise atleast a portion of the desired gene. In some embodiments, the DNA iscleaved at specific sites using various restriction enzymes. In somealternative embodiments, DNAse is used in the presence of manganese tofragment the DNA, or the DNA is physically sheared (e.g., bysonication). The linear DNA fragments created are then be separatedaccording to size and amplified by standard techniques, including butnot limited to, agarose and polyacrylamide gel electrophoresis, PCR andcolumn chromatography.

Once nucleic acid fragments are generated, identification of thespecific DNA fragment encoding a protease may be accomplished in anumber of ways. For example, in some embodiments, a proteolytichydrolyzing enzyme encoding the asp gene or its specific RNA, or afragment thereof, such as a probe or primer, is isolated, labeled, andthen used in hybridization assays well known to those in the art, todetect a generated gene (See e.g., Benton and Davis, Science 196:180[1977]; and Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72:3961[1975]). In preferred embodiments, DNA fragments sharing substantialsequence similarity to the probe hybridize under medium to highstringency.

In some preferred embodiments, amplification is accomplished using PCR,as known in the art. In some preferred embodiments, a nucleic acidsequence of at least about 4 nucleotides and as many as about 60nucleotides from SEQ ID NOS:1-5, (i.e., fragments), preferably about 12to 30 nucleotides, and more preferably about 25 nucleotides are used inany suitable combinations as PCR primer. These same fragments also finduse as probes in hybridization and product detection methods.

In some embodiments, isolation of nucleic acid constructs of theinvention from a cDNA or genomic library utilizes PCR with usingdegenerate oligonucleotide primers prepared on the basis of the aminoacid sequence of the protein. The primers can be of any segment length,for example at least 4, at least 5, at least 8, at least 15, at least20, nucleotides in length.

In view of the above, it will be appreciated that the polynucleotidesequences provided herein and based on the polynucleotide sequencesprovided in SEQ ID NOS:1-5 are useful for obtaining identical orhomologous fragments of polynucleotides from other species, andparticularly from bacteria that encode enzymes having the serineprotease activity expressed by protease 69B4. Additional sequences areprovided in U.S. patent application Ser. No. 10/576,331.

IV. Multiple Mutation Variants of Serine Proteases of the PresentInvention

As indicated herein, in particularly preferred embodiments, the presentinvention provides multiple mutation variants of serine protease. Insome most particularly preferred embodiments, these variants exhibitimproved performance as compared to the parent (e.g., wild-type)protease. In some of these most particularly preferred embodiments, thevariants have improved wash performance, LAS stability, and/orproteolytic activity. Thus, these variants find use in numerousapplications, including but not limited to dishwashing detergents,laundry detergents, and surface cleaning detergents.

V. Expression and Recovery of Serine Proteases of the Present Invention

Any suitable means for expression and recovery of the serine proteasesof the present invention find use herein. Indeed, those of skill in theart know many methods suitable for cloning a Cellulomonas-derivedpolypeptide having proteolytic activity, as well as an additional enzyme(e.g., a second peptide having proteolytic activity, such as a protease,cellulase, mannanase, or amylase, etc.). Numerous methods are also knownin the art for introducing at least one (e.g., multiple) copies of thepolynucleotide(s) encoding the enzyme(s) of the present invention inconjunction with any additional sequences desired, into the genes orgenome of host cells.

In general, standard procedures for cloning of genes and introducingexogenous proteases encoding regions (including multiple copies of theexogenous encoding regions) into said genes find use in obtaining aCellulomonas 69B4 protease derivative or homologue thereof. Indeed, thepresent Specification, including the Examples provides such teaching.However, additional methods known in the art are also suitable (Seee.g., Sambrook et al. supra (1989); Ausubel et al., supra [1995]; andHarwood and Cutting, (eds.) Molecular Biological Methods for Bacillus,”John Wiley and Sons, [1990]; and WO 96/34946).

In some preferred embodiments, the polynucleotide sequences of thepresent invention are expressed by operatively linking them to anexpression control sequence in an appropriate expression vector andemployed by that expression vector to transform an appropriate hostaccording to techniques well established in the art. In someembodiments, the polypeptides produced on expression of the DNAsequences of this invention are isolated from the fermentation of cellcultures and purified in a variety of ways according to well-establishedtechniques in the art. Those of skill in the art are capable ofselecting the most appropriate isolation and purification techniques.

More particularly, the present invention provides constructs, vectorscomprising polynucleotides described herein, host cells transformed withsuch vectors, proteases expressed by such host cells, expression methodsand systems for the production of serine protease enzymes derived frommicroorganisms, in particular, members of the Micrococcineae, includingbut not limited to Cellulomonas species. In some embodiments, thepolynucleotide(s) encoding serine protease(s) are used to producerecombinant host cells suitable for the expression of the serineprotease(s). In some preferred embodiments, the expression hosts arecapable of producing the protease(s) in commercially viable quantities.Additional details are provided in U.S. patent application Ser. No.10/576,331.

VI. Recombinant Vectors and Host Cells

As indicated above, in some embodiments, the present invention providesvectors comprising the aforementioned polynucleotides. In someembodiments, the vectors (i.e., constructs) of the invention encodingthe protease are of genomic origin (e.g., prepared though use of agenomic library and screening for DNA sequences coding for all or partof the protease by hybridization using synthetic oligonucleotide probesin accordance with standard techniques). These vectors are described ingreater detail in U.S. patent application Ser. No. 10/576,331.

As indicated above, in some embodiments, the present invention alsoprovides host cells transformed with the vectors described above.Additional host cells are described in greater detail in U.S. patentapplication Ser. No. 10/576,331.

VII. Applications for Serine Protease Enzymes

As described in greater detail herein, the proteases of the presentinvention have important characteristics that make them very suitablefor certain applications. For example, the proteases of the presentinvention have enhanced thermal stability. In some embodiments, theenzymes also exhibit enhanced oxidative stability, and enhanced chelatorstability, as compared to some currently used proteases. Thus, theseproteases find use in cleaning compositions. Indeed, under certain washconditions, the present proteases exhibit comparative or enhanced washperformance as compared with currently used subtilisin proteases. Thus,it is contemplated that the cleaning and/or enzyme compositions of thepresent invention will be provided in a variety of cleaningcompositions. In some embodiments, the proteases of the presentinvention are utilized in the same manner as subtilisin proteases (i.e.,proteases currently in use). Thus, the present proteases find use invarious cleaning compositions, as well as animal feed applications,leather processing (e.g., bating), protein hydrolysis, and in textileuses. The identified proteases also find use in personal careapplications.

Thus, the proteases of the present invention find use in a number ofindustrial applications, in particular within the cleaning,disinfecting, animal feed, and textile/leather industries. In someembodiments, the protease(s) of the present invention are combined withdetergents, builders, bleaching agents and other conventionalingredients to produce a variety of novel cleaning compositions usefulin the laundry and other cleaning arts such as, for example, laundrydetergents (both powdered and liquid), laundry pre-soaks, all fabricbleaches, automatic dishwashing detergents (both liquid and powdered),household cleaners, particularly bar and liquid soap applications, anddrain openers. In addition, the proteases find use in the cleaning ofcontact lenses, as well as other items, by contacting such materialswith an aqueous solution of the cleaning composition. In addition thesenaturally occurring proteases can be used, for example in peptidehydrolysis, waste treatment, textile applications, medical devicecleaning, biofilm removal and as fusion-cleavage enzymes in proteinproduction, etc. The composition of these products is not critical tothe present invention, as long as the protease(s) maintain theirfunction in the setting used. In some embodiments, the compositions arereadily prepared by combining a cleaning effective amount of theprotease or an enzyme composition comprising the protease enzymepreparation with the conventional components of such compositions intheir art recognized amounts.

The proteases of the present invention find particular use in thecleaning industry, including, but not limited to laundry and dishdetergents. These applications place enzymes under various environmentalstresses. The proteases of the present invention provide advantages overmany currently used enzymes, due to their stability under variousconditions.

Indeed, there are a variety of wash conditions including varyingdetergent formulations, wash water volumes, wash water temperatures, andlengths of wash time, to which proteases involved in washing areexposed. In addition, detergent formulations used in differentgeographical areas have different concentrations of their relevantcomponents present in the wash water. For example, a European detergenttypically has about 4500-5000 ppm of detergent components in the washwater, while a Japanese detergent typically has approximately 667 ppm ofdetergent components in the wash water. In North America, particularlythe United States, detergents typically have about 975 ppm of detergentcomponents present in the wash water.

A low detergent concentration system includes detergents where less thanabout 800 ppm of detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of detergent components are present in thewash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water. Brazil typicallyhas approximately 1500 ppm of detergent components present in the washwater.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500-5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Asmentioned above, Brazil typically has approximately 1500 ppm ofdetergent components present in the wash water. However, other high sudsphosphate builder detergent geographies, not limited to other LatinAmerican countries, may have high detergent concentration systems up toabout 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 800 ppm of detergent composition (“lowdetergent concentration geographies”), for example about 667 ppm inJapan, to between about 800 ppm to about 2000 ppm (“medium detergentconcentration geographies”), for example about 975 ppm in U.S. and about1500 ppm in Brazil, to greater than about 2000 ppm (“high detergentconcentration geographies”), for example about 4500 ppm to about 5000ppm in Europe and about 6000 ppm in high suds phosphate buildergeographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 975 ppm of detergent within the wash solutionabout 62.79 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

As a further example, different geographies use different washtemperatures. The temperature of the wash water in Japan is typicallyless than that used in Europe. For example, the temperature of the washwater in North America and Japan can be between 10 and 30° C. (e.g.,about 20° C.), whereas the temperature of wash water in Europe istypically between 30 and 60° C. (e.g., about 40° C.).

As a further example, different geographies typically have differentwater hardness. Water hardness is usually described in terms of thegrains per gallon mixed Ca²⁺/Mg²⁺. Hardness is a measure of the amountof calcium (Ca²⁺) and magnesium (Mg²⁺) in the water. Most water in theUnited States is hard, but the degree of hardness varies. Moderatelyhard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts permillion (parts per million converted to grains per U.S. gallon is ppm #divided by 17.1 equals grains per gallon) of hardness minerals (See,Table 13-1).

European water hardness is typically greater than 10.5 (e.g., 10.5-20.0)grains per gallon mixed Ca²⁺/Mg²⁺ (e.g., about 15 grains per gallonmixed Ca²⁺/Mg²⁺). North American water hardness is typically greaterthan Japanese water hardness, but less than European water hardness. Forexample, North American water hardness can be between 3 to 10 grains,3-8 grains or about 6 grains. Japanese water hardness is typically lowerthan North American water hardness, usually less than 4, for example 3grains per gallon mixed Ca²⁺/Mg²⁺.

Accordingly, in some embodiments, the present invention providesproteases that show surprising wash performance in at least one set ofwash conditions (e.g., water temperature, water hardness, and/ordetergent concentration). In some embodiments, the proteases of thepresent invention are comparable in wash performance to subtilisinproteases. In some embodiments, the proteases of the present inventionexhibit enhanced wash performance as compared to subtilisin proteases.Thus, in some preferred embodiments of the present invention, theproteases provided herein exhibit enhanced oxidative stability, enhancedthermal stability, and/or enhanced chelator stability.

In some preferred embodiments, the present invention provides the ASPprotease, as well as homologues and variants of the protease. Inparticularly preferred embodiments, the ASP variants comprise multiplesubstitutions in the wild-type ASP protease sequence. These proteasesfind use in any applications in which it is desired to clean proteinbased stains from textiles or fabrics.

In some embodiments, the cleaning compositions of the present inventionare formulated as hand and machine laundry detergent compositionsincluding laundry additive compositions, and compositions suitable foruse in the pretreatment of stained fabrics, rinse-added fabric softenercompositions, and compositions for use in general household hard surfacecleaning operations, as well as dishwashing operations. Those in the artare familiar with different formulations which can be used as cleaningcompositions. In preferred embodiments, the proteases of the presentinvention comprise comparative or enhanced performance in detergentcompositions (i.e., as compared to other proteases). In someembodiments, cleaning performance is evaluated by comparing theproteases of the present invention with subtilisin proteases in variouscleaning assays that utilize enzyme-sensitive stains such as egg, grass,blood, milk, etc., in standard methods. Indeed, those in the art arefamiliar with the spectrophotometric and other analytical methodologiesused to assess detergent performance under standard wash cycleconditions.

Assays that find use in the present invention include, but are notlimited to those described in WO 99/34011, and U.S. Pat. No. 6,605,458(See e.g., Example 3). In U.S. Pat. No. 6,605,458, at Example 3, adetergent dose of 3.0 g/l at pH10.5, wash time 15 minutes, at 15 C,water hardness of 6° dH, 10 nM enzyme concentration in 150 ml glassbeakers with stirring rod, 5 textile pieces (phi 2.5 cm) in 50 ml, EMPA117 test material from Center for Test Materials Holland are used. Themeasurement of reflectance “R” on the test material was done at 460 nmusing a Macbeth ColorEye 7000 photometer. Additional methods areprovided in the Examples herein. Thus, these methods also find use inthe present invention.

The addition of proteases of the invention to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe present compositions, as long as the pH is within the range setforth herein, and the temperature is below the described protease'sdenaturing temperature. In addition, proteases of the present inventionfind use in cleaning compositions that do not include detergents, againeither alone or in combination with builders and stabilizers.

When used in cleaning compositions or detergents, oxidative stability isa further consideration. Thus, in some applications, the stability isenhanced, diminished, or comparable to subtilisin proteases as desiredfor various uses. In some preferred embodiments, enhanced oxidativestability is desired. Some of the proteases of the present inventionfind particular use in such applications.

When used in cleaning compositions or detergents, thermal stability is afurther consideration. Thus, in some applications, the stability isenhanced, diminished, or comparable to subtilisin proteases as desiredfor various uses. In some preferred embodiments, enhancedthermostability is desired. Some of the proteases of the presentinvention find particular use in such applications.

When used in cleaning compositions or detergents, chelator stability isa further consideration. Thus, in some applications, the stability isenhanced, diminished, or comparable to subtilisin proteases as desiredfor various uses. In some preferred embodiments, enhanced chelatorstability is desired. Some of the proteases of the present inventionfind particular use in such applications.

In some embodiments of the present invention, naturally occurringproteases are provided which exhibit modified enzymatic activity atdifferent pHs when compared to subtilisin proteases. A pH-activityprofile is a plot of pH against enzyme activity and may be constructedas described in the Examples and/or by methods known in the art. In someembodiments, it is desired to obtain naturally occurring proteases withbroader profiles (i.e., those having greater activity at range of pHsthan a comparable subtilisin protease). In other embodiments, theenzymes have no significantly greater activity at any pH, or naturallyoccurring homologues with sharper profiles (i.e., those having enhancedactivity when compared to subtilisin proteases at a given pH, and lesseractivity elsewhere). Thus, in various embodiments, the proteases of thepresent invention have differing pH optima and/or ranges. It is notintended that the present invention be limited to any specific pH or pHrange.

In some embodiments of the present invention, the cleaning compositionscomprise, proteases of the present invention at a level from 0.00001% to10% of 69B4 and/or other proteases of the present invention by weight ofthe composition and the balance (e.g., 99.999% to 90.0%) comprisingcleaning adjunct materials by weight of composition. In other aspects ofthe present invention, the cleaning compositions of the presentinvention comprise, the 69B4 and/or other proteases at a level of0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% 69B4 or otherprotease of the present invention by weight of the composition and thebalance of the cleaning composition (e.g., 99.9999% to 90.0%, 99.999% to98%, 99.995% to 99.5% by weight) comprising cleaning adjunct materials.

In some embodiments, preferred cleaning compositions, in addition to theprotease preparation of the invention, comprise one or more additionalenzymes or enzyme derivatives which provide cleaning performance and/orfabric care benefits. Such enzymes include, but are not limited to otherproteases, lipases, cutinases, amylases, cellulases, peroxidases,oxidases (e.g. laccases), and/or mannanases.

Any other protease suitable for use in alkaline solutions finds use inthe compositions of the present invention. Suitable proteases includethose of animal, vegetable or microbial origin. In particularlypreferred embodiments, microbial proteases are used. In someembodiments, chemically or genetically modified mutants are included. Insome embodiments, the protease is a serine protease, preferably analkaline microbial protease or a trypsin-like protease. Examples ofalkaline proteases include subtilisins, especially those derived fromBacillus (e.g., subtilisin, lentus, amyloliquefaciens, subtilisinCarlsberg, subtilisin 309, subtilisin 147 and subtilisin 168).Additional examples include those mutant proteases described in U.S.Pat. Nos. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, allof which are incorporated herein by reference. Additional proteaseexamples include, but are not limited to trypsin (e.g., of porcine orbovine origin), and the hprotease described in WO 89/06270. Preferredcommercially available protease enzymes include those sold under thetrade names MAXATASE®, MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®,PROPERASE®, PURAFECT® and PURAFECT® OXP (Genencor), those sold under thetrade names ALCALASE®, SAVINASE®, PRIMASE®, DURAZYM™, RELASE® andESPERASE® (Novozymes); and those sold under the trade name BLAP™ (HenkelKommanditgesellschaft auf Aktien, Duesseldorf, Germany. Variousproteases are described in WO95/23221, WO 92/21760, and U.S. Pat. Nos.5,801,039, 5,340,735, 5,500,364, 5,855,625. An additional BPN′ variant(“BPN′-var 1” and “BPN-variant 1”; as referred to herein) is describedin US RE 34,606. An additional GG36-variant (“GG36-var.1” and“GG36-variant 1”; as referred to herein) is described in U.S. Pat. Nos.5,955,340 and 5,700,676. A further GG36-variant is described in U.S.Pat. Nos. 6,312,936 and 6,482,628. In one aspect of the presentinvention, the cleaning compositions of the present invention compriseadditional protease enzymes at a level from 0.00001% to 10% ofadditional protease by weight of the composition and 99.999% to 90.0% ofcleaning adjunct materials by weight of composition. In otherembodiments of the present invention, the cleaning compositions of thepresent invention also comprise, proteases at a level of 0.0001% to 10%,0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% 69B4 protease (or itshomologues or variants) by weight of the composition and the balance ofthe cleaning composition (e.g., 99.9999% to 90.0%, 99.999% to 98%,99.995% to 99.5% by weight) comprising cleaning adjunct materials.

In addition, any lipase suitable for use in alkaline solutions finds usein the present invention. Suitable lipases include, but are not limitedto those of bacterial or fungal origin. Chemically or geneticallymodified mutants are encompassed by the present invention. Examples ofuseful lipases include Humicola lanuginosa lipase (See e.g., EP 258 068,and EP 305 216), Rhizomucor miehei lipase (See e.g., EP 238 023),Candida lipase, such as C. antarctica lipase (e.g., the C. antarcticalipase A or B; See e.g., EP 214 761), a Pseudomonas lipase such as P.alcaligenes and P. pseudoalcaligenes lipase (See e.g., EP 218 272), P.cepacia lipase (See e.g., EP 331 376), P. stutzeri lipase (See e.g., GB1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B. subtilislipase [Dartois et al., Biochem. Biophys. Acta 1131:253-260 [1993]); B.stearothermophilus lipase [See e.g., JP 64/744992]; and B. pumiluslipase [See e.g., WO 91/16422]).

Furthermore, a number of cloned lipases find use in some embodiments ofthe present invention, including but not limited to Penicilliumcamembertii lipase (See, Yamaguchi et al., Gene 103:61-67 [1991]),Geotricum candidum lipase (See, Schimada et al., J. Biochem.,106:383-388 [1989]), and various Rhizopus lipases such as R. delemarlipase (See, Hass et al., Gene 109:117-113 [1991]), a R. niveus lipase(Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 [1992]) and R.oryzae lipase.

Other types of lipolytic enzymes such as cutinases also find use in someembodiments of the present invention, including but not limited to thecutinase derived from Pseudomonas mendocina (See, WO 88/09367), orcutinase derived from Fusarium solani pisi (See, WO 90/09446).

Additional suitable lipases include commercially available lipases suchas M1 LIPASE™, LUMA FAST™, and LIPOMAX™ (Genencor); LIPOLASE® andLIPOLASE® ULTRA (Novozymes); and LIPASE P™ “Amano” (Amano PharmaceuticalCo. Ltd., Japan).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise lipases at a level from0.00001% to 10% of additional lipase by weight of the composition andthe balance of cleaning adjunct materials by weight of composition. Inother aspects of the present invention, the cleaning compositions of thepresent invention also comprise, lipases at a level of 0.0001% to 10%,0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% lipase by weight of thecomposition.

Any amylase (alpha and/or beta) suitable for use in alkaline solutionsalso find use in some embodiments of the present invention. Suitableamylases include, but are not limited to those of bacterial or fungalorigin. Chemically or genetically modified mutants are included in someembodiments. Amylases that find use in the present invention, include,but are not limited to α-amylases obtained from B. licheniformis (Seee.g., GB 1,296,839). Commercially available amylases that find use inthe present invention include, but are not limited to DURAMYL®,TERMAMYL®, FUNGAMYL®, STAINZYME®, and BAN™ (Novozymes) and RAPIDASE®,and MAXAMYL® P (Genencor International). In some embodiments of thepresent invention, the cleaning compositions of the present inventionfurther comprise amylases at a level from 0.00001% to 10% of additionalamylase by weight of the composition and the balance of cleaning adjunctmaterials by weight of composition. In other aspects of the presentinvention, the cleaning compositions of the present invention alsocomprise, amylases at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to2%, 0.005% to 0.5% amylase by weight of the composition.

Any cellulase suitable for use in alkaline solutions find use inembodiments of the present invention. Suitable cellulases include, butare not limited to those of bacterial or fungal origin. Chemically orgenetically modified mutants are included in some embodiments. Suitablecellulases include, but are not limited to Humicola insolens cellulases(See e.g., U.S. Pat. No. 4,435,307). Especially suitable cellulases arethe cellulases having color care benefits (See e.g., EP 0 495 257).

Commercially available cellulases that find use in the present include,but are not limited to CELLUZYME® (Novozymes), and KAC-500(B)™ (KaoCorporation). In some embodiments, cellulases are incorporated asportions or fragments of mature wild-type or variant cellulases, whereina portion of the N-terminus is deleted (See e.g., U.S. Pat. No.5,874,276).

In some embodiments, the cleaning compositions of the present inventioncan further comprise cellulases at a level from 0.00001% to 10% ofadditional cellulase by weight of the composition and the balance ofcleaning adjunct materials by weight of composition. In other aspects ofthe present invention, the cleaning compositions of the presentinvention also comprise cellulases at a level of 0.0001% to 10%, 0.001%to 5%, 0.001% to 2%, 0.005% to 0.5% cellulase by weight of thecomposition.

Any mannanases suitable for use in detergent compositions and oralkaline solutions find use in the present invention. Suitablemannanases include, but are not limited to those of bacterial or fungalorigin. Chemically or genetically modified mutants are included in someembodiments. Various mannanases are known which find use in the presentinvention (See e.g., U.S. Pat. No. 6,566,114, U.S. Pat. No. 6,602,842,and U.S. Pat. No. 6,440,991, all of which are incorporated herein byreference).

In some embodiments, the cleaning compositions of the present inventioncan further comprise mannanases at a level from 0.00001% to 10% ofadditional mannanase by weight of the composition and the balance ofcleaning adjunct materials by weight of composition. In other aspects ofthe present invention, the cleaning compositions of the presentinvention also comprise, mannanases at a level of 0.0001% to 10%, 0.001%to 5%, 0.001% to 2%, 0.005% to 0.5% mannanases by weight of thecomposition.

In some embodiments, peroxidases are used in combination with hydrogenperoxide or a source thereof (e.g., a percarbonate, perborate orpersulfate). In alternative embodiments, oxidases are used incombination with oxygen. Both types of enzymes are used for “solutionbleaching” (i.e., to prevent transfer of a textile dye from a dyedfabric to another fabric when the fabrics are washed together in a washliquor), preferably together with an enhancing agent (See e.g., WO94/12621 and WO 95/01426). Suitable peroxidases/oxidases include, butare not limited to those of plant, bacterial or fungal origin.Chemically or genetically modified mutants are included in someembodiments.

In some embodiments, the cleaning compositions of the present inventioncan further comprise peroxidase and/or oxidase enzymes at a level from0.00001% to 10% of additional peroxidase and/or oxidase by weight of thecomposition and the balance of cleaning adjunct materials by weight ofcomposition. In other aspects of the present invention, the cleaningcompositions of the present invention also comprise, peroxidase and/oroxidase enzymes at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to2%, 0.005% to 0.5% peroxidase and/or oxidase enzymes by weight of thecomposition.

Mixtures of the above mentioned enzymes are encompassed herein, inparticular a mixture of a the 69B4 enzyme, one or more additionalproteases, at least one amylase, at least one lipase, at least onemannanase, and/or at least one cellulase. Indeed, it is contemplatedthat various mixtures of these enzymes will find use in the presentinvention.

It is contemplated that the varying levels of the protease and one ormore additional enzymes may both independently range to 10%, the balanceof the cleaning composition being cleaning adjunct materials. Thespecific selection of cleaning adjunct materials are readily made byconsidering the surface, item, or fabric to be cleaned, and the desiredform of the composition for the cleaning conditions during use (e.g.,through the wash detergent use).

Examples of suitable cleaning adjunct materials include, but are notlimited to, surfactants, builders, bleaches, bleach activators, bleachcatalysts, other enzymes, enzyme stabilizing systems, chelants, opticalbrighteners, soil release polymers, dye transfer agents, dispersants,suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes,photoactivators, fluorescers, fabric conditioners, hydrolyzablesurfactants, preservatives, anti-oxidants, anti-shrinkage agents,anti-wrinkle agents, germicides, fungicides, color speckles, silvercare,anti-tarnish and/or anti-corrosion agents, alkalinity sources,solubilizing agents, carriers, processing aids, pigments, and pH controlagents (See e.g., U.S. Pat. Nos. 6,610,642, 6,605,458, 5,705,464,5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of whichare incorporated herein by reference). Embodiments of specific cleaningcomposition materials are exemplified in detail below.

If the cleaning adjunct materials are not compatible with the proteasesof the present invention in the cleaning compositions, then suitablemethods of keeping the cleaning adjunct materials and the protease(s)separated (i.e., not in contact with each other) until combination ofthe two components is appropriate are used. Such separation methodsinclude any suitable method known in the art (e.g., gelcaps,encapulation, tablets, physical separation, etc.).

Preferably an effective amount of one or more protease(s) providedherein are included in compositions useful for cleaning a variety ofsurfaces in need of proteinaceous stain removal. Such cleaningcompositions include cleaning compositions for such applications ascleaning hard surfaces, fabrics, and dishes. Indeed, in someembodiments, the present invention provides fabric cleaningcompositions, while in other embodiments, the present invention providesnon-fabric cleaning compositions. Notably, the present invention alsoprovides cleaning compositions suitable for personal care, includingoral care (including dentrifices, toothpastes, mouthwashes, etc., aswell as denture cleaning compositions), skin, and hair cleaningcompositions. It is intended that the present invention encompassdetergent compositions in any form (i.e., liquid, granular, bar,semi-solid, gels, emulsions, tablets, capsules, etc.).

By way of example, several cleaning compositions wherein the protease ofthe present invention find use are described in greater detail below. Inembodiments in which the cleaning compositions of the present inventionare formulated as compositions suitable for use in laundry machinewashing method(s), the compositions of the present invention preferablycontain at least one surfactant and at least one builder compound, aswell as one or more cleaning adjunct materials preferably selected fromorganic polymeric compounds, bleaching agents, additional enzymes, sudssuppressors, dispersants, lime-soap dispersants, soil suspension andanti-redeposition agents and corrosion inhibitors. In some embodiments,laundry compositions also contain softening agents (i.e., as additionalcleaning adjunct materials).

The compositions of the present invention also find use detergentadditive products in solid or liquid form. Such additive products areintended to supplement and/or boost the performance of conventionaldetergent compositions and can be added at any stage of the cleaningprocess.

In embodiments formulated as compositions for use in manual dishwashingmethods, the compositions of the invention preferably contain at leastone surfactant and preferably at least one additional cleaning adjunctmaterial selected from organic polymeric compounds, suds enhancingagents, group II metal ions, solvents, hydrotropes and additionalenzymes.

In some embodiments, the density of the laundry detergent compositionsherein ranges from 400 to 1200 g/liter, while in other embodiments, itranges from 500 to 950 g/liter of composition measured at 20° C.

In some embodiments, various cleaning compositions such as thoseprovided in U.S. Pat. No. 6,605,458 find use with the proteases of thepresent invention. Thus, in some embodiments, the compositionscomprising at least one protease of the present invention is a compactgranular fabric cleaning composition, while in other embodiments, thecomposition is a granular fabric cleaning composition useful in thelaundering of colored fabrics, in further embodiments, the compositionis a granular fabric cleaning composition which provides softeningthrough the wash capacity, in additional embodiments, the composition isa heavy duty liquid fabric cleaning composition.

In some embodiments, the compositions comprising at least one proteaseof the present invention are fabric cleaning compositions such as thosedescribed in U.S. Pat. No. 6,610,642 and U.S. Pat. No. 6,376,450. Inaddition, the proteases of the present invention find use in granularlaundry detergent compositions of particular utility under European orJapanese washing conditions (See e.g., U.S. Pat. No. 6,610,642).

In alternative embodiments, the present invention provides hard surfacecleaning compositions comprising at least one protease provided herein.Thus, in some embodiments, the compositions comprising at least oneprotease of the present invention is a hard surface cleaning compositionsuch as those described in U.S. Pat. No. 6,610,642, U.S. Pat. No.6,376,450, and U.S. Pat. No. 6,376,450.

In yet further embodiments, the present invention provides dishwashingcompositions comprising at least one protease provided herein. Thus, insome embodiments, the compositions comprising at least one protease ofthe present invention is a hard surface cleaning composition such asthose in U.S. Pat. No. 6,610,642, and U.S. Pat. No. 6,376,450.

In still further embodiments, the present invention provides dishwashingcompositions comprising at least one protease provided herein. Thus, insome embodiments, the compositions comprising at least one protease ofthe present invention comprise oral care compositions such as those inU.S. Pat. No. 6,376,450, and U.S. Pat. No. 6,376,450.

The formulations and descriptions of the compounds and cleaning adjunctmaterials contained in the aforementioned U.S. Pat. Nos. 6,376,450;6,605,458; 6,605,458; and 6,610,642 are expressly incorporated byreference herein. Still further examples are set forth in the Examplesbelow.

Still further, the present invention provides compositions and methodsfor the production of a food or animal feed, characterized in thatprotease according to the invention is mixed with food or animal feed.In some embodiments, the protease is added as a dry product beforeprocessing, while in other embodiments it is added as a liquid before orafter processing. In some embodiments, in which a dry powder is used,the enzyme is diluted as a liquid onto a dry carrier such as milledgrain. The proteases of the present invention find use as components ofanimal feeds and/or additives such as those described in U.S. Pat. No.5,612,055, U.S. Pat. No. 5,314,692, and U.S. Pat. No. 5,147,642, all ofwhich are hereby incorporated by reference.

The enzyme feed additive according to the present invention is suitablefor preparation in a number of methods. For example, in someembodiments, it is prepared simply by mixing different enzymes havingthe appropriate activities to produce an enzyme mix. In someembodiments, this enzyme mix is mixed directly with a feed, while inother embodiments, it is impregnated onto a cereal-based carriermaterial such as milled wheat, maize or soya flour. The presentinvention also encompasses these impregnated carriers, as they find useas enzyme feed additives.

In some alternative embodiments, a cereal-based carrier (e.g., milledwheat or maize) is impregnated either simultaneously or sequentiallywith enzymes having the appropriate activities. For example, in someembodiments, a milled wheat carrier is first sprayed with a xylanase,secondly with a protease, and optionally with β-glucanase. The presentinvention also encompasses these impregnated carriers, as they find useas enzyme feed additives. In preferred embodiments, these impregnatedcarriers comprise at least one protease of the present invention.

In some embodiments, the feed additive of the present invention isdirectly mixed with the animal feed, while in alternative embodiments,it is mixed with one or more other feed additives such as a vitamin feedadditive, a mineral feed additive, and/or an amino acid feed additive.The resulting feed additive including several different types ofcomponents is then mixed in an appropriate amount with the feed.

In some preferred embodiments, the feed additive of the presentinvention, including cereal-based carriers is normally mixed in amountsof 0.01-50 g per kilogram of feed, more preferably 0.1-10 g/kilogram,and most preferably about 1 g/kilogram.

In alternative embodiments, the enzyme feed additive of the presentinvention involves construction of recombinant microorganisms thatproduces the desired enzyme(s) in the desired relative amounts. In someembodiments, this is accomplished by increasing the copy number of thegene encoding at least one protease of the present invention, and/or byusing a suitably strong promoter operatively linked to thepolynucleotide encoding the protease(s). In further embodiments, therecombinant microorganism strain has certain enzyme activities deleted(e.g., cellulases, endoglucanases, etc.), as desired.

In additional embodiments, the enzyme feed additives provided by thepresent invention also include other enzymes, including but not limitedto at least one xylanase, α-amylase, glucoamylase, pectinase, mannanase,α-galactosidase, phytase, and/or lipase. In some embodiments, theenzymes having the desired activities are mixed with the xylanase andprotease either before impregnating these on a cereal-based carrier oralternatively such enzymes are impregnated simultaneously orsequentially on such a cereal-based carrier. The carrier is then in turnmixed with a cereal-based feed to prepare the final feed. In alternativeembodiments, the enzyme feed additive is formulated as a solution of theindividual enzyme activities and then mixed with a feed materialpre-formed as pellets or as a mash.

In still further embodiments, the enzyme feed additive is included inanimals' diets by incorporating it into a second (i.e., different) feedor the animals' drinking water. Accordingly, it is not essential thatthe enzyme mix provided by the present invention be incorporated intothe cereal-based feed itself, although such incorporation forms aparticularly preferred embodiment of the present invention. The ratio ofthe units of xylanase activity per g of the feed additive to the unitsof protease activity per g of the feed additive is preferably1:0.001-1,000, more preferably 1:0.01-100, and most preferably 1:0.1-10.As indicated above, the enzyme mix provided by the present inventionpreferably finds use as a feed additive in the preparation of acereal-based feed.

In some embodiments, the cereal-based feed comprises at least 25% byweight, or more preferably at least 35% by weight, wheat or maize or acombination of both of these cereals. The feed further comprises aprotease (i.e., at least one protease of the present invention) in suchan amount that the feed includes a protease in such an amount that thefeed includes 100-100,000 units of protease activity per kg.

Cereal-based feeds provided the present invention according to thepresent invention find use as feed for a variety of non-human animals,including poultry (e.g., turkeys, geese, ducks, chickens, etc.),livestock (e.g., pigs, sheep, cattle, goats, etc.), and companionanimals (e.g., horses, dogs, cats, rabbits, mice, etc.). The feeds areparticularly suitable for poultry and pigs, and in particular broilerchickens.

The present invention also provides compositions for the treatment oftextiles that include at least one of the proteases of the presentinvention. In some embodiments, at least one protease of the presentinvention is a component of compositions suitable for the treatment ofsilk or wool (See e.g., U.S. RE Pat. No. 216,034, EP 134,267, U.S. Pat.No. 4,533,359, and EP 344,259).

In addition, the proteases of the present invention find use in avariety of applications where it is desirable to separate phosphorousfrom phytate. Accordingly, the present invention also provides methodsproducing wool or animal hair material with improved properties. In somepreferred embodiments, these methods comprise the steps of pretreatingwool, wool fibres or animal hair material in a process selected from thegroup consisting of plasma treatment processes and the Delhey process;and subjecting the pretreated wool or animal hair material to atreatment with a proteolytic enzyme (e.g., at least one protease of thepresent invention) in an amount effective for improving the properties.In some embodiments, the proteolytic enzyme treatment occurs prior tothe plasma treatment, while in other embodiments, it occurs after theplasma treatment. In some further embodiments, it is conducted as aseparate step, while in other embodiments, it is conducted incombination with the scouring or the dyeing of the wool or animal hairmaterial. In additional embodiments, at least one surfactant and/or atleast one softener is present during the enzyme treatment step, while inother embodiments, the surfactant(s) and/or softener(s) are incorporatedin a separate step wherein the wool or animal hair material is subjectedto a softening treatment.

In some embodiments, the compositions of the present invention find usein methods for shrink-proofing wool fibers (See e.g., JP 4-327274). Insome embodiments, the compositions are used in methods forshrink-proofing treatment of wool fibers by subjecting the fibers to alow-temperature plasma treatment, followed by treatment with ashrink-proofing resin such as a block-urethane resin, polyamideepochlorohydrin resin, glyoxalic resin, ethylene-urea resin or acrylateresin, and then treatment with a weight reducing proteolytic enzyme forobtaining a softening effect). In some embodiments, the plasma treatmentstep is a low-temperature treatment, preferably a corona dischargetreatment or a glow discharge treatment.

In some embodiments, the low-temperature plasma treatment is carried outby using a gas, preferably a gas selected from the group consisting ofair, oxygen, nitrogen, ammonia, helium, or argon. Conventionally, air isused but it may be advantageous to use any of the other indicatedgasses.

Preferably, the low-temperature plasma treatment is carried out at apressure between about 0.1 torr and 5 ton for from about 2 seconds toabout 300 seconds, preferably for about 5 seconds to about 100 seconds,more preferably from about 5 seconds to about 30 seconds.

As indicated above, the present invention finds use in conjunction withmethods such as the Delhey process (See e.g., DE-A-43 32 692). In thisprocess, the wool is treated in an aqueous solution of hydrogen peroxidein the presence of soluble wolframate, optionally followed by treatmentin a solution or dispersion of synthetic polymers, for improving theanti-felting properties of the wool. In this method, the wool is treatedin an aqueous solution of hydrogen peroxide (0.1-35% (w/w), preferably2-10% (w/w)), in the presence of a 2-60% (w/w), preferably 8-20% (w/w)of a catalyst (preferably Na₂WO₄), and in the presence of a nonionicwetting agent. Preferably, the treatment is carried out at pH 8-11, androom temperature. The treatment time depends on the concentrations ofhydrogen peroxide and catalyst, but is preferably 2 minutes or less.After the oxidative treatment, the wool is rinsed with water. Forremoval of residual hydrogen peroxide, and optionally for additionalbleaching, the wool is further treated in acidic solutions of reducingagents (e.g., sulfites, phosphites etc.).

In some embodiments, the enzyme treatment step carried out for betweenabout 1 minute and about 120 minutes. This step is preferably carriedout at a temperature of between about 20° C. and about 60° C., morepreferably between about 30° C. and about 50° C. Alternatively, the woolis soaked in or padded with an aqueous enzyme solution and thensubjected to steaming at a conventional temperature and pressure,typically for about 30 seconds to about 3 minutes. In some preferredembodiments, the proteolytic enzyme treatment is carried out in anacidic or neutral or alkaline medium which may include a buffer.

In alternative embodiments, the enzyme treatment step is conducted inthe presence of one or more conventional anionic, non-ionic (e.g.,Dobanol; Henkel AG) or cationic surfactants. An example of a usefulnonionic surfactant is Dobanol (from Henkel AG). In further embodiments,the wool or animal hair material is subjected to an ultrasoundtreatment, either prior to or simultaneous with the treatment with aproteolytic enzyme. In some preferred embodiments, the ultrasoundtreatment is carried out at a temperature of about 50° C. for about 5minutes. In some preferred embodiments, the amount of proteolytic enzymeused in the enzyme treatment step is between about 0.2 w/w % and about10 w/w %, based on the weight of the wool or animal hair material. Insome embodiments, in order to the number of treatment steps, the enzymetreatment is carried out during dyeing and/or scouring of the wool oranimal hair material, simply by adding the protease to the dyeing,rinsing and/or scouring bath. In some embodiments, enzyme treatment iscarried out after the plasma treatment but in other embodiments, the twotreatment steps are carried out in the opposite order.

Softeners conventionally used on wool are usually cationic softeners,either organic cationic softeners or silicone based products, butanionic or non-inoc softeners are also useful. Examples of usefulsofteners include, but are not limited to polyethylene softeners andsilicone softeners (i.e., dimethyl polysiloxanes (silicone oils)),H-polysiloxanes, silicone elastomers, aminofunctional dimethylpolysiloxanes, aminofunctional silicone elastomers, and epoxyfunctionaldimethyl polysiloxanes, and organic cationic softeners (e.g. alkylquarternary ammonium derivatives).

In additional embodiments, the present invention provides compositionsfor the treatment of an animal hide that includes at least one proteaseof the present invention. In some embodiments, the proteases of thepresent invention find use in compositions for treatment of animal hide,such as those described in WO 03/00865 (Insect Biotech Co., Taejeon-Si,Korea). In additional embodiments, the present invention providesmethods for processing hides and/or skins into leather comprisingenzymatic treatment of the hide or skin with the protease of the presentinvention (See e.g., WO 96/11285). In additional embodiments, thepresent invention provides compositions for the treatment of an animalskin or hide into leather that includes at least one protease of thepresent invention.

Hides and skins are usually received in the tanneries in the form ofsalted or dried raw hides or skins. The processing of hides or skinsinto leather comprises several different process steps including thesteps of soaking, unhairing and bating. These steps constitute the wetprocessing and are performed in the beamhouse. Enzymatic treatmentutilizing the proteases of the present invention are applicable at anytime during the process involved in the processing of leather. However,proteases are usually employed during the wet processing (i.e., duringsoaking, unhairing and/or bating). Thus, in some preferred embodiments,the enzymatic treatment with at least one of the proteases of thepresent invention occurs during the wet processing stage.

In some embodiments, the soaking processes of the present invention areperformed under conventional soaking conditions (e.g., at a pH in therange pH 6.0-11). In some preferred embodiments, the range is pH7.0-10.0. In alternative embodiments, the temperature is in the range of20-30° C., while in other embodiments it is preferably in the range24-28° C. In yet further embodiments, the reaction time is in the range2-24 hours, while preferred range is 4-16 hours. In additionalembodiments, tensides and/or preservatives are provided as desired.

The second phase of the bating step usually commences with the additionof the bate itself. In some embodiments, the enzymatic treatment takesplace during bating. In some preferred embodiments, the enzymatictreatment takes place during bating, after the deliming phase. In someembodiments, the bating process of the presents invention is performedusing conventional conditions (e.g., at a pH in the range pH 6.0-9.0).In some preferred embodiments, the pH range is 6.0 to 8.5. In furtherembodiments, the temperature is in the range of 20-30° C., while inpreferred embodiments, the temperature is in the range of 25-28° C. Insome embodiments, the reaction time is in the range of 20-90 minutes,while in other embodiments, it is in the range 40-80 minutes. Processesfor the manufacture of leather are well-known to those skilled in theart (See e.g., WO 94/069429 WO 90/1121189, U.S. Pat. No. 3,840,433, EP505920, GB 2233665, and U.S. Pat. No. 3,986,926, all of which are hereinincorporated by reference).

In further embodiments, the present invention provides bates comprisingat least one protease of the present invention. A bate is an agent or anenzyme-containing preparation comprising the chemically activeingredients for use in beamhouse processes, in particular in the batingstep of a process for the manufacture of leather. In some embodiments,the present invention provides bates comprising protease and suitableexcipients. In some embodiments, agents including, but not limited tochemicals known and used in the art, e.g. diluents, emulgators, delimersand carriers. In some embodiments, the bate comprising at least oneprotease of the present invention is formulated as known in the art (Seee.g., GB-A2250289, WO 96/11285, and EP 0784703).

In some embodiments, the bate of the present invention contains from0.00005 to 0.01 g of active protease per g of bate, while in otherembodiments, the bate contains from 0.0002 to 0.004 g of active proteaseper g of bate.

Thus, the proteases of the present invention find use in numerousapplications and settings.

Experimental

The present invention is described in further detail in the followingExamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein. The following Examples are offered toillustrate, but not to limit the claimed invention

In the experimental disclosure which follows, the followingabbreviations apply: PI (proteinase inhibitor), ppm (parts per million);M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol(moles); mmol (millimoles); μmmol (micromoles); nmol (nanomoles); gm(grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters);ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm(millimeters); μm (micrometers); nm (nanometers); U (units); V (volts);MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) andhr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient);ND (not done); NA (not applicable); rpm (revolutions per minute); H₂O(water); dH₂O (deionized water); (HCl (hydrochloric acid); aa (aminoacid); by (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copyor complementary DNA); DNA (deoxyribonucleic acid); ssDNA (singlestranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotidetriphosphate); RNA (ribonucleic acid); MgCl₂ (magnesium chloride); NaCl(sodium chloride); w/v (weight to volume); v/v (volume to volume); g(gravity); OD (optical density); Dulbecco's phosphate buffered solution(DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl,2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto Tryptone, 24 g/l glycerol,2.31 g/l KH₂PO₄, and 12.54 g/l K₂HPO₄); OD₂₈₀ (optical density at 280nm); OD₆₀₀ (optical density at 600 nm); A₄₀₅ (absorbance at 405 nm);Vmax (the maximum initial velocity of an enzyme catalyzed reaction);PAGE (polyacrylamide gel electrophoresis); PBS (phosphate bufferedsaline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST(PBS+0.25% TWEEN® 20); PEG (polyethylene glycol); PCR (polymerase chainreaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecylsulfate); Tris(tris(hydroxymethyl)aminomethane); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine(N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino)ethane-sulfonic acid); TAPS(3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS(3-(cyclo-hexylamino)-propanesulfonic acid; DMSO (dimethyl sulfoxide);DTT (1,4-dithio-DL-threitol); SA (sinapinic acid(s,5-dimethoxy-4-hydroxy cinnamic acid); TCA (trichloroacetic acid);Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP(Tris[2-carboxyethyl]phosphine); Ci (Curies); mCi (milliCuries); μCi(microcuries); HPLC (high pressure liquid chromatography); RP-HPLC(reverse phase high pressure liquid chromatography); TLC (thin layerchromatography); MALDI-TOF (matrix-assisted laserdesorption/ionization-time of flight); Ts (tosyl); Bn (benzyl); Ph(phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (Thermus aquaticusDNA polymerase); Klenow (DNA polymerase I large (Klenow) fragment); EGTA(ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid);EDTA (ethylenediaminetetracetic acid); bla (β-lactamase orampicillin-resistance gene); HDL (high density liquid); MJ Research (MJResearch, Reno, Nev.); Baseclear (Baseclear BV, Inc., Leiden, theNetherlands); PerSeptive (PerSeptive Biosystems, Framingham, Mass.);ThermoFinnigan (ThermoFinnigan, San Jose, Calif.); Argo (ArgoBioAnalytica, Morris Plains, N.J.); Seitz EKS (SeitzSchenk FiltersystemsGmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East Hills, N.Y.); EMPATestmaterialien AG (EMPA Testmaterialien AG, St. Gallen-Winkeln,Switzerland); Warwick Equest (Warwick Equest Limited, Durham, UK);Minolta (Konica Minolta, Inc., Japan); United States Testing (UnitedStates Testing Co, Inc, Hoboken, N.J.); Spectrum (Spectrum Laboratories,Dominguez Rancho, Calif.); Molecular Structure (Molecular StructureCorp., Woodlands, Tex.); Accelrys (Accelrys, Inc., San Diego, Calif.);Chemical Computing (Chemical Computing Corp., Montreal, Canada); NewBrunswick (New Brunswick Scientific, Co., Edison, N.J.); CFT (Center forTest Materials, Vlaardingen, the Netherlands); Procter & Gamble (Procter& Gamble, Inc., Cincinnati, Ohio); GE Healthcare (GE Healthcare,Chalfont St. Giles, United Kingdom); DNA2.0 (DNA2.0, Menlo Park,Calif.); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (MegazymeInternational Ireland Ltd., Bray Business Park, Bray, Co., Wicklow,Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP Kelco,Wilmington, Del.); Corning (Corning Life Sciences, Corning, N.Y.); (NEN(NEN Life Science Products, Boston, Mass.); Pharma AS (Pharma AS, Oslo,Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis,Lewisville, Tex.); ATCC (American Type Culture Collection, Rockville,Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma ChemicalCo., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Piscataway, N.J.);NCBI (National Center for Biotechnology Information); Applied Biosystems(Applied Biosystems, Foster City, Calif.); BD Biosciences and/orClontech (BD Biosciences CLONTECH Laboratories, Palo Alto, Calif.);Operon Technologies (Operon Technologies, Inc., Alameda, Calif.); MWGBiotech (MWG Biotech, High Point, N.C.); Oligos Etc (Oligos Etc. Inc,Wilsonville, Oreg.); Bachem (Bachem Bioscience, Inc., King of Prussia,Pa.); Difco (Difco Laboratories, Detroit, Mich.); Mediatech (Mediatech,Herndon, Va.; Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.); Oxoid (Oxoid Inc., Ogdensburg, N.Y.); Worthington (WorthingtonBiochemical Corp., Freehold, N.J.); GIBCO BRL or Gibco BRL (LifeTechnologies, Inc., Gaithersburg, Md.); Millipore (Millipore, Billerica,Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (InvitrogenCorp., San Diego, Calif.); NEB (New England Biolabs, Beverly, Mass.);Sigma (Sigma Chemical Co., St. Louis, Mo.); Pierce (PierceBiotechnology, Rockford, Ill.); Takara (Takara Bio Inc. Otsu, Japan);Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science,Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.); Biodesign(Biodesign Intl., Saco, Me.); Aptagen (Aptagen, Inc., Herndon, Va.);Sorvall (Sorvall brand, from Kendro Laboratory Products, Asheville,N.C.); Molecular Devices (Molecular Devices, Corp., Sunnyvale, Calif.);R&D Systems (R&D Systems, Minneapolis, Minn.); Stratagene (StratageneCloning Systems, La Jolla, Calif.); Marsh (Marsh Biosciences, Rochester,N.Y.); Geneart (Geneart GmbH, Regensburg, Germany); Bio-Tek (Bio-TekInstruments, Winooski, Vt.); (Biacore (Biacore, Inc., Piscataway, N.J.);PeproTech (PeproTech, Rocky Hill, N.J.); SynPep (SynPep, Dublin,Calif.); New Objective (New Objective brand; Scientific InstrumentServices, Inc., Ringoes, N.J.); Waters (Waters, Inc., Milford, Mass.);Matrix Science (Matrix Science, Boston, Mass.); Dionex (Dionex, Corp.,Sunnyvale, Calif.); Monsanto (Monsanto Co., St. Louis, Mo.); Wintershall(Wintershall AG, Kassel, Germany); BASF (BASF Co., Florham Park, N.J.);Huntsman (Huntsman Petrochemical Corp., Salt Lake City, Utah); Enichem(Enichem Iberica, Barcelona, Spain); Fluka Chemie AG (Fluka Chemie AG,Buchs, Switzerland); Gist-Brocades (Gist-Brocades, Nev., Delft, theNetherlands); Dow Corning (Dow Corning Corp., Midland, Mich.); andMicrosoft (Microsoft, Inc., Redmond, Wash.).

The wild-type serine protease used in the following Examples isdescribed in detail in US04/39006 and US04/39066, both of which areherein incorporated by reference in their entirety. In addition, Example2 of U.S. patent application Ser. No. 10/576,331 provides details forthe production of 69B4 protease from the Gram-positive alkaliphilicbacterium 69B4. As indicated throughout the present application, thispriority application is incorporated by reference herein in itsentirety.

Example 1 Assays

In the following Examples, various assays were used, such as proteindeterminations, application-based tests, and stability-based tests. Forease in reading, the following assays are set forth below and referredto in the respective Examples. Any deviations from the protocolsprovided below in any of the experiments performed during thedevelopment of the present invention are indicated in the Examples.

Some of the detergents used in the following Examples had the followingcompositions. In Compositions I and II, the balance (to 100%) isperfume/dye and/or water. The pH of these compositions was from about 5to about 7 for Composition I, and about 7.5 to about 8.5 Composition II.In Composition III, the balance (to 100%) comprised of water and/or theminors perfume, dye, brightener/SRPI/sodiumcarboxymethylcellulose/photobleach/MgSo₄/PVPVI/suds suppressor/highmolecular PEG/clay.

DETERGENT COMPOSITIONS Composition Composition I II LAS 24.0 8.0C₁₂-C₁₅AE_(1.8)S — 11.0 C₈-C₁₀ propyl dimethyl 2.0 2.0 amine C₁₂-C₁₄alkyl dimethyl — — amine oxide C₁₂-C₁₅AS — 7.0 CFAA — 4.0 C₁₂-C₁₄ Fattyalcohol 12.0 1.0 ethoxylate C₁₂-C₁₈ Fatty acid 3.0 4.0 Citric acid(anhydrous) 6.0 3.0 DETPMP — 1.0 Monoethanolamine 5.0 5.0 Sodiumhydroxide — 1.0 1N HCl aqueous #1 — solution Propanediol 12.7 10.Ethanol 1.8 5.4 DTPA 0.5 0.4 Pectin Lyase — 0.005 Lipase 0.1 — Amylase0.001 — Cellulase — 0.0002 Protease A — — Aldose Oxidase — — DETBCHD —0.01 SRP1 0.5 0.3 Boric acid 2.4 2.8 Sodium xylene — — sulfonate DC3225C 1.0 1.0 2-butyl-octanol 0.03 0.03 Brightener 1 0.12 0.08

Detergent Composition III C₁₄-C₁₅AS or sodium 3.0 tallow alkyl sulfateLAS 8.0 C₁₂-C₁₅AE₃S 1.0 C₁₂-C₁₅E₅ or E₃ 5.0 QAS — Zeolite A 11.0  SKS-6(dry add) 9.0 MA/AA 2.0 AA — 3Na Citrate 2H₂O — Citric Acid (Anhydrous)1.5 DTPA — EDDS 0.5 HEDP 0.2 PB1 — Percarbonate 3.8 NOBS — NACA OBS 2.0TAED 2.0 BB1  0.34 BB2 — Anhydrous Na Carbonate 8.0 Sulfate 2.0 Silicate— Protease B — Protease C — Lipase — Amylase — Cellulase — Pectin Lyase 0.001 Aldose Oxidase  0.05 PAAC —A. TCA Assay for Protein Content Determination in 96-well MicrotiterPlates

This assay was started using filtered culture supernatant frommicrotiter plates grown 4 days at 33° C. with shaking at 230 RPM andhumidified aeration. A fresh 96-well flat bottom plate was used for theassay. First, 100 μL/well of 0.25 N HCl were placed in the wells. Then,50 μL filtered culture broth were added to the wells. The lightscattering/absorbance at 405 nm (use 5 sec mixing mode in the platereader) was then determined, in order to provide the “blank” reading.

For the test, 100 μL/well 15% (w/v) TCA was placed in the plates andincubated between 5 and 30 min at room temperature. The lightscattering/absorbance at 405 nm (use 5 sec mixing mode in the platereader) was then determined.

The calculations were performed by subtracting the blank (i.e., no TCA)from the test reading with TCA. If desired, a standard curve can becreated by calibrating the TCA readings with AAPF assays of clones withknown conversion factors. However, the TCA results are linear withrespect to protein concentration from 50 to 500 ppm and can thus beplotted directly against enzyme performance for the purpose of choosinggood-performing variants.

B. Suc-AAPF-pNA Assay of Proteases in 96-well Microtiter Plates

In this assay system, the reagent solutions used were:

1. 100 mM Tris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris buffer)

2. 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl₂ and 0.005%TWEEN®-80 (Tris buffer)

3. 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma:S-7388)

To prepare suc-AAPF-pNA working solution, 1 ml AAPF stock was added to100 ml Tris buffer and mixed well for at least 10 seconds.

The assay was performed by adding 10 μl of diluted protease solution toeach well, followed by the addition (quickly) of 190 μl 1 mg/mlAAPF-working solution. The solutions were mixed for 5 sec., and theabsorbance change was read at 410 nm in an MTP reader, at 25° C. Theprotease activity was expressed as AU (activity=ΔOD·min⁻¹.ml⁻¹).

C. Keratin Hydrolysis Assay

In this assay system, the chemical and reagent solutions used were:

-   Keratin ICN 902111-   Detergent 1.6 g. detergent was dissolved in 1000 ml water (pH=8.2)    0.6 ml. CaCl₂/MgCl₂ of 10,000 gpg was also added, as well as 1190 mg    HEPES, giving a hardness and buffer strength of 6 gpg and 5 mM    respectively. The pH was adjusted to 8.2 with NaOH.-   Picrylsulfonic acid (TNBS) Sigma P-2297 (5% solution in water)-   Reagent A 45.4 g Na₂B₄O₇.10 H2O (Merck 6308) and 15 ml of 4N NaOH    were dissolved together to a final volume of 1000 ml (by heating if    needed)-   Reagent B 35.2 g NaH₂PO₄.1H₂O (Merck 6346) and 0.6 g Na₂SO₃    (Merck 6657) were dissolved together to a final volume of 1000 ml.    Method:

Prior to the incubations, keratin was sieved on a 100 μm sieve in smallportions at a time. Then, 10 g of the <100 μm keratin was stirred indetergent solution for at least 20 minutes at room temperature withregular adjustment of the pH to 8.2. Finally, the suspension wascentrifuged for 20 minutes at room temperature (Sorvall, GSA rotor,13,000 rpm). This procedure was then repeated. Finally, the wet sedimentwas suspended in detergent to a total volume of 200 ml., and thesuspension was kept stirred during pipetting. Prior to incubation,microtiter plates (MTPs) were filled with 200 μl substrate per well witha Biohit multichannel pipette and 1200 μl tip (6 dispenses of 200 μl anddispensed as fast as possible to avoid settling of keratin in the tips).Then, 10 μl of the filtered culture was added to the substratecontaining MTPs. The plates were covered with tape, placed in anincubator and incubated at 20° C. for 3 hours at 350 rpm (Innova 4330[New Brunswick]). Following incubation, the plates were centrifuged for3 minutes at 3000 rpm (iSigma 6K 15 centrifuge). About 15 minutes beforeremoval of the 1^(st) plate from the incubator, the TNBS reagent wasprepared by mixing 1 ml TNBS solution per 50 ml of reagent A.

MTPs were filled with 60 μl TNBS reagent A per well. From the incubatedplates, 10 μl was transferred to the MTPs with TNBS reagent A. Theplates were covered with tape and shaken for 20 minutes in a benchshaker (BMG Thermostar) at room temperature and 500 rpm. Finally, 200 μlof reagent B was added to the wells, mixed for 1 minute on a shaker, andthe absorbance at 405 nm was measured with the MTP-reader.

Calculation of the Keratin Hydrolyzing Activity:

The obtained absorbance value was corrected for the blank value(substrate without enzyme). The resulting absorbance provides a measurefor the hydrolytic activity. For each sample (variant) the performanceindex was calculated. The performance index compares the performance ofthe variant (actual value) and the standard enzyme (theoretical value)at the same protein concentration. In addition, the theoretical valuescan be calculated, using the parameters of the Langmuir equation of thestandard enzyme. A performance index (PI) that is greater than 1 (PI>1)identifies a better variant (as compared to the standard [e.g.,wild-type]), while a PI of 1 (PI=1) identifies a variant that performsthe same as the standard, and a PI that is less than 1 (PI<1) identifiesa variant that performs worse than the standard. Thus, the PI identifieswinners, as well as variants that are less desirable for use undercertain circumstances.

D. Microswatch Assay for Testing Protease Performance

All of the detergents used in these assays did not contain enzymes.

Detergent Preparations:

1. Cold Water Liquid Detergent (US Conditions):

Milli-Q water was adjusted to 6 gpg water hardness (Ca/Mg=3/1), 1.60 g/ldetergent was added, and the detergent solution was stirred vigorouslyfor at least 15 minutes.

Then, 5 mM Hepes buffer was added and the pH adjusted to 8.2. Thedetergent was filtered before use in the assay through a 0.22 μm filter(e.g. Nalgene top bottle filter).

2. Low pH Liquid Detergent (US Conditions):

Milli-Q water was adjusted to 6 gpg water hardness (Ca/Mg=3/1), 1.60 g/ldetergent TIDE®-LVJ-1 or TIDE® 2005) or 1.50 g/l detergent (TIDE®-SNOW)was added, and the detergent solution stirred vigorously for at least 15minutes. The pH was adjusted to 6.0 using 1N NaOH. solution. Thedetergent was filtered before use in the assay through a 0.22 μm filter(e.g. Nalgene top bottle filter).

Microswatches:

Microswatches of ¼″ circular diameter were ordered and delivered by CFTVlaardingen. The microswatches were pretreated using the fixation methoddescribed below. Single microswatches were placed in each well of a96-well microtiter plate vertically to expose the whole surface area(i.e., not flat on the bottom of the well).

“3K” Swatch Fixation:

This particular swatch fixation was done at room temperature. Howeverthe amount of 30% H₂O₂ added is 10× more than in the superfixed swatchfixation method i.e., conducted at 60° C., used with Europeanconditions). Bubble formation (frothing) will be visible and thereforeit is necessary to use a bigger beaker to account for this. First, 8liters of distilled water were placed in a 10 L beaker, and 80 ml of 30%hydrogen peroxide added. The water and peroxide were mixed well with aladle. Then, 40 pieces of EMPA 116 swatches were spread into a fanbefore adding into the solution to ensure uniform fixation. The swatcheswere swirled in the solution (using the ladle) for 30 minutes,continuously for the first five minutes and occasionally for theremaining 25 minutes. The solution was discarded and the swatches wererinsed 6 times with approximately 6 liters of distilled water each time.The swatches were placed on top of paper towels to dry. The air-driedswatches were punched using a ¼″ circular die on an expulsion press. Asingle microswatch was placed vertically into each well of a 96-wellmicrotiter plate to expose the whole surface area (i.e., not flat on thebottom of the well).

Enzyme Samples:

The enzyme samples were tested at appropriate concentrations for therespective geography, and diluted in 10 mM NaCl, 0.005% TWEEN®-80solution.

Test Method:

The incubator was set at the desired temperature: 20° C. for cold waterliquid conditions or 30° C. for low-pH liquid conditions. The pretreatedand precut swatches were placed into the wells of a 96-well MTP, asdescribed above. The enzyme samples were diluted, if needed, in 10 mMNaCl, 0.005% TWEEN®-80 to 20× the desired concentration. The desireddetergent solutions were prepared as described above. Then, 190 μl ofdetergent solution were added to each well of the MTP. To this mixture,10 μl of enzyme solution were added to each well (to provide a totalvolume to 200 μl/well). The MTP was sealed with a plate sealer andplaced in an incubator for 60 minutes, with agitation at 350 rpm.Following incubation under the appropriate conditions, 100 μl ofsolution from each well were removed and placed into a fresh MTP. Thenew MTP containing 100 μl of solution/well was read at 405 nm in a MTPreader. Blank controls, as well as a control containing a microswatchand detergent but no enzyme were also included. The stock solution wasused at a concentration of 15,000 gpg (Ca/Mg 3:1 (1.92 M Ca²⁺=282.3 g/LCaCl₂.2H₂O; 0.64 M Mg²⁺=30.1 g/L MgCl₂.6H₂O).

TABLE 1-1 Detergent Composition and Incubation Conditions in the μSwatchAssay. Enzyme Reference Dosage Temp. Detergent Enzyme Detergent WaterHardness [ppm] (° C.) Swatch Cold Water ASP 1.5 or 1.6 g/l 6 gpg -Ca/Mg: 0.3-4 20° 3K Liquid Detergent 3/1 Low pH ASP 1.6 g/l 6 gpg -0.5-4 30° 3K Liquid Detergent Ca/Mg: 3/1 DetergentCalculation of the BMI Performance:

The obtained absorbance value was corrected for the blank value(obtained after incubation of microswatches in the absence of enzyme).The resulting absorbance was a measure for the hydrolytic activity. Foreach sample (variant) the performance index was calculated. Theperformance index compares the performance of the variant (actual value)and the standard enzyme (theoretical value) at the same proteinconcentration. In addition, the theoretical values can be calculated,using the parameters of the Langmuir equation of the standard enzyme. Aperformance index (PI) that is greater than 1 (PI>1) identifies a bettervariant (as compared to the standard [e.g., wild-type]), while a PI of 1(PI=1) identifies a variant that performs the same as the standard, anda PI that is less than 1 (PI<1) identifies a variant that performs worsethan the standard.

Thus, the PI identifies winners, as well as variants that are lessdesirable for use under certain circumstances.

D. Dimethylcasein Hydrolysis Assay (96 wells)

In this assay system, the chemical and reagent solutions used were:

-   Dimethylcasein (DMC): Sigma C-9801-   TWEEN®-80: Sigma P-8074-   PIPES buffer (free acid): Sigma P-1851; 15.1 g is dissolved in about    960 ml water; pH is adjusted: to 7.0 with 4N NaOH, 1 ml 5% TWEEN®-80    is added and the volume brought up to 1000 ml. The final    concentration of PIPES and TWEEN®-80 is 50 mM and 0.005%    respectively.-   Picrylsulfonic acid (TNBS): Sigma P-2297 (5% solution in water)-   Reagent A: 45.4 g Na₂B₄O₇.10 H2O (Merck 6308) and 15 ml of 4N NaOH    are dissolved together to a final volume of 1000 ml (by heating if    needed)-   Reagent B: 35.2 g NaH₂PO₄.1H₂O (Merck 6346) and 0.6 g Na₂SO₃    (Merck 6657) are dissolved together to a final volume of 1000 ml.    Method:

To prepare the substrate, 4 g DMC were dissolved in 400 ml PIPES buffer.The filtered culture supernatants were diluted with PIPES buffer; thefinal concentration of the controls in the growth plate was 20 ppm.Then, 10 μl of each diluted supernatant were added to 200 μl substratein the wells of a MTP. The MTP plate was covered with tape, shaken for afew seconds and placed in an oven at 37° C. for 2 hours withoutagitation.

About 15 minutes before removal of the 1^(St) plate from the oven, theTNBS reagent was prepared by mixing 1 ml TNBS solution per 50 ml ofreagent A. MTPs were filled with 60 μl TNBS reagent A per well. Theincubated plates were shaken for a few seconds, after which 10 μl weretransferred to the MTPs with TNBS reagent A. The plates were coveredwith tape and shaken for 20 minutes in a bench shaker (BMG Thermostar)at room temperature and 500 rpm. Finally, 200 μl reagent B were added tothe wells, mixed for 1 minute on a shaker, and the absorbance at 405 nmwas determined using an MTP-reader.

Calculation of Dimethylcasein Hydrolyzing Activity:

The obtained absorbance value was corrected for the blank value(substrate without enzyme). The resulting absorbance is a measure forthe hydrolytic activity. The (arbitrary) specific activity of a samplewas calculated by dividing the absorbance and the determined proteinconcentration.

E. Thermostability Assay

This assay is based on the dimethylcasein hydrolysis, before and afterheating of the buffered culture supernatant. The same chemical andreagent solutions were used as described in the dimethylcaseinhydrolysis assay.

Method:

The filtered culture supernatants were diluted to 20 ppm in PIPES buffer(based on the concentration of the controls in the growth plates).First, 10 ul of each diluted enzyme sample was taken to determine theinitial activity in the dimethylcasein assay and treated as describedbelow. Then, 50 μl of each diluted supernatant were placed in the emptywells of a MTP. The MTP plate was incubated in an iEMS incubator/shakerHT (Thermo Labsystems) for 90 minutes at 60° C. and 400 rpm. The plateswere cooled on ice for 5 minutes. Then, 10 μl of the solution was addedto a fresh MTP containing 200 μl dimethylcasein substrate/well todetermine the final activity after incubation. This MTP was covered withtape, shaken for a few seconds and placed in an oven at 37° C. for 2hours without agitation. The same detection method as used for the DMChydrolysis assay was used.

Calculation of Thermostability:

The residual activity of a sample was expressed as the ratio of thefinal absorbance and the initial absorbance, both corrected for blanks.

F. LAS Stability Assay

LAS stability was measured after incubation of the test protease in thepresence of 0.06% LAS (dodecylbenzenesulfonate sodium), and the residualactivity was determined using the AAPF assay.

Reagents:

Dodecylbenzenesulfonate, Sodium salt (=LAS): Sigma D-2525

TWEEN®-80: Sigma P-8074

TRIS buffer (free acid): Sigma T-1378); 6.35 g is dissolved in about 960ml water; pH is adjusted to 8.2 with 4N HCl. Final concentration of TRISis 52.5 mM.

LAS stock solution: Prepare a 10.5% LAS solution in MQ water (=10.5 gper 100 ml MQ)

TRIS buffer-100 mM/pH 8.6 (100 mM Tris/0.005% Tween80)

TRIS-Ca buffer, pH 8.6 (100 mM Tris/10 mM CaCl₂/0.005% Tween80)

Hardware:

Flat bottom MTPs: Costar (#9017)

Biomek FX

ASYS Multipipettor

Spectramax MTP Reader

iEMS Incubator/Shaker

Innova 4330 Incubator/Shaker

Biohit multichannel pipette

BMG Thermostar Shaker

Method:

A 0.063% LAS solution was prepared in 52.5 mM Tris buffer pH 8.2. TheAAPF working solution was prepared by adding 1 ml of 100 mg/ml AAPFstock solution (in DMSO) to 100 ml (100 mM) TRIS buffer, pH 8.6. Todilute the supernatants, flat-bottomed plates were filled with dilutionbuffer and an aliquot of the supernatant was added and mixed well. Thedilution ratio depended on the concentration of the ASP-controls in thegrowth plates (AAPF activity). The desired protein concentration was 80ppm.

Ten μl of the diluted supernatant were added to 190 μl 0.063% LASbuffer/well. The MTP was covered with tape, shaken for a few seconds andplaced in an incubator (Innova 4230) at 25° or 35° C., for 60 minutes at200 rpm agitation. The initial activity (t=10 minutes) was determinedafter 10 minutes of incubation by transferring 10 μl of the mixture ineach well to a fresh MTP containing 190 μl AAPF work solution. Thesesolutions were mixed well and the AAPF activity was measured using a MTPReader (20 readings in 5 minutes and 25° C.).

The final activity (t=60 minutes) was determined by removing another 10μl of solution from the incubating plate after 60 minutes of incubation.The AAPF activity was then determined as described above. Thecalculations were performed as follows:the % Residual Activity was [t−60 value]*100/[t−10 value].

Example 2 ASP Protease Production in B. subtilis

In this Example, experiments conducted to produce 69B4 protease (alsoreferred to herein as “ASP,” “Asp,” and “ASP protease,” and “Aspprotease”) in B. subtilis are described. In this Example, thetransformation of plasmid pHPLT-ASP-C1-2 (See, FIG. 1) into B. subtilisis described. Transformation was performed as known in the art (Seee.g., WO 02/14490, incorporated herein by reference). To optimize ASPexpression in B. subtilis, a synthetic DNA sequence was produced byDNA2.0, and utilized in these expression experiments. The DNA sequence(synthetic ASP DNA sequence) provided below, with codon usage adaptedfor Bacillus species, encodes the wild type ASP precursor protein:

(SEQ ID NO: 10)ATGACACCACGAACTGTCACAAGAGCTCTGGCTGTGGCAACAGCAGCTGCTACACTCTTGGCTGGGGGTATGGCAGCACAAGCTAACGAACCGGCTCCTCCAGGATCTGCATCAGCCCCTCCACGATTAGCTGAAAAACTTGACCCTGACTTACTTGAAGCAATGGAACGCGATCTGGGGTTAGATGCAGAGGAAGCAGCTGCAACGTTAGCTTTTCAGCATGACGCAGCTGAAACGGGAGAGGCTCTTGCTGAGGAACTCGACGAAGATTTCGCGGGCACGTGGGTTGAAGATGATGTGCTGTATGTTGCAACCACTGATGAAGATGCTGTTGAAGAAGTCGAAGGCGAAGGAGCAACTGCTGTGACTGTTGAGCATTCTCTTGCTGATTTAGAGGCGTGGAAGACGGTTTTGGATGCTGCGCTGGAGGGTCATGATGATGTGCCTACGTGGTACGTCGACGTGCCTACGAATTCGGTAGTCGTTGCTGTAAAGGCAGGAGCGCAGGATGTAGCTGCAGGACTTGTGGAAGGCGCTGATGTGCCATCAGATGCGGTCACTTTTGTAGAAACGGACGAAACGCCTAGAACGATG TTCGACGTAATTGGAGGCAACGCATATACTATTGGCGGCCGGTCTAGATGTTCTATCGGATTCGCAGTAAACGGTGGCTTCATTACTGCCGGTCACTGCGGAAGAACAGGAGCCACTACTGCCAATCCGACTGGCACATTTGCAGGTAGCTCGTTTCCGGGAAATGATTATGCATTCGTCCGAACAGGGGCAGGAGTAAATTTGCTTGCCCAAGTCAATAACTACTCGGGCGGCAGAGTCCAAGTAGCAGGACATACGGCCGCACCAGTTGGATCTGCTGTATGCCGCTCAGGTAGCACTACAGGTTGGCATTGCGGAACTATCACGGCGCTGAATTCGTCTGTCACGTATCCAGAGGGAACAGTCCGAGGACTTATCCGCACGACGGTTTGTGCCGAACCAGGTGATAGCGGAGGTAGCCTTTTAGCGGGAAATCAAGCCCAAGGTGTCACGTCAGGTGGTTCTGGAAATTGTCGGACGGGGGGAACAACATTCTTTCAACCAGTCAACCCGATTTTGCAGGCTTACGGCCTGAGAATGATTACGACTGACTCTGGAAGTTCCCCT GCTCCAGCACCTACATCATGTACAGGCTACGCAAGAACGTTCACAGGAACCCTCGCAGCAGGAAGAGCAGCAGCTCAACCGAACGGTAGCTATGTTCAGGTCAACCGGAGCGGTACACATTCCGTCTGTCTCAATGGACCTAGCGGTGCGGACTTTGATTTGTATGTGCAGCGATGGAATGGCAGTAGCTGGGTAACCGTCGCTCAATCGACATCGCCGGGAAGCAATGAAACCATTACGTACCGCGGAAATGCTGGATATTATCGCTACGTGGTTAACGCTGCGTCAGGATCAGGAGCTTACACAATGGGACTCACCCTCCCCTGA 

In the above sequence, bold indicates the DNA that encodes the matureprotease, standard font indicates the leader sequence, and the underlineindicates the N-terminal and C-terminal prosequences.

Expression of the Synthetic ASP Gene

Asp expression cassettes were constructed in the pXX-KpnI (See, FIG. 2)vector and subsequently cloned into the pHPLT vector (See, FIG. 3) forexpression of ASP in B. subtilis. pXX-KpnI is a pUC based vector withthe aprE promoter (B. subtilis) driving expression, a cat gene, and aduplicate aprE promoter for amplification of the copy number in B.subtilis. The bla gene allows selective growth in E. coli. The KpnI,introduced in the ribosomal binding site, downstream of the aprEpromoter region, together with the HindIII site enables cloning of Aspexpression cassettes in pXX-KpnI. pHPLT-EBS2c2, a derivative of pHPLT(Solingen et al., Extremophiles 5:333-341 [2001]), contains thethermostable amylase LAT promoter (P_(LAT)) of Bacillus licheniformis,followed by XbaI and HpaI restriction sites for cloning ASP expressionconstructs.

The Asp expression cassette was cloned in the pXX-KpnI vector containingDNA encoding a hybrid signal peptide constructed of 5 subtilisin AprEN-terminal signal peptide amino acids fused to the 25 Asp C-terminalsignal peptide amino acids (MRSKKRTVTRALAVATAAATLLAGGMAAQA; SEQ IDNO:11). The Asp expression cassette cloned in the pXX-KpnI vector wastransformed into E. coli (Electromax DH10B, Invitrogen, Cat. No.12033-015). The primers and cloning strategy used are provided in Table2-1. Subsequently, the expression cassettes were cloned from thesevectors and introduced in the pHPLT expression vector for transformationinto a B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32,ΔamyE::(xylR,pxylA-comK) strain. The primers and cloning strategy forASP expression cassettes cloning in pHPLT are provided in Table 2-2.Transformation into B. subtilis was performed as described in WO02/14490, incorporated herein by reference.

TABLE 2-1 ASP in pXX-KpnI and p2JM103-DNNDPI Restriction ASP C-Sites Used Vector Signal Terminal DNA Host for Construct Peptideprosequence Primers Template Vector Cloning pXX- MRSKKRTVT NotASP-PreCross-I-FW ASP pXX- KpnI and ASP-4 RALAVATAA incorporatedTCATGCAGGGTACCATG synthetic KpnI HindIII ATLLAGGMA AGAAGCAAGAAGCGAA DNAAQA (SEQ CTGTCACAAGAGCTCTG sequence ID NO: 11) GCT (SEQ ID NO: 12)ASP-syntc-mature-RV GTGTGCAAGCTTTCAAG GGGAACTTCCAGAGTCAGTC (SEQ ID NO: 13)

TABLE 2-2 ASP Expression Cassettes in pHPLT Restriction Vector DNASites Used Construct Primers Template Host Vector for Cloning pHPLT-ASP-Cross-1&2-FW pXX-ASP-4 PHPLT- NheI x SmaI ASP-TGAGCTGCTAGCAAAAGGAGAGGG EBS2c2 (XbaI x C1-2 TAAAGAATGAGAAGCAAGAAG HpaI)(SEQ ID NO: 14) pHPLT-ASPmat-RV CATGCATCCCGGGTTAAGGGGAACTTCCAGAGTCAGTC (SEQ ID NO: 15)

Primers were obtained from MWG and Invitrogen. Invitrogen Platinum TaqDNA polymerase High Fidelity (Cat. No. 11304-029) was used for PCRamplification (0.2 μM primers, 25 up to 30 cycles) according to theInvitrogen's protocol. Ligase reactions of ASP expression cassettes andhost vectors were completed by using Invitrogen T4 DNA Ligase (Cat. No.15224-025), utilizing Invitrogen's protocol as recommended for generalcloning of cohesive ends).

Expression of the asp gene was investigated in a B. subtilis strain(ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xylR,pxylA-comK.) Theplasmid pHPLT-ASP-C1-2 (See, Table 2-2, and FIG. 1), was transformedinto B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32,ΔamyE::(xylR,pxylA-comK). Transformation was performed as known in theart (See e.g., WO 02/14490, incorporated herein by reference).

Selective growth of B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32,ΔamyE::(xylR,pxylA-comK) transformants harboring the pHPLT-ASP-C1-2vector was performed in shake flasks containing 25 ml Synthetic MaxataseMedium (SMM), with 0.97 g/l CaCl₂.6H₂O instead of 0.5 g/l CaCl₂ (See,U.S. Pat. No. 5,324,653, herein incorporated by reference) with 20 mg/Lneomycin. This growth resulted in the production of secreted ASPprotease with proteolytic activity. Gel analysis was performed usingNuPage Novex 10% Bis-Tris gels (Invitrogen, Cat. No. NP0301BOX). Toprepare samples for analysis, 2 volumes of supernatant were mixed with 1volume 1M HCl, 1 volume 4xLDS sample buffer (Invitrogen, Cat. No.NP0007), and 1% PMSF (20 mg/ml) and subsequently heated for 10 minutesat 70° C. Then, 25 μL of each sample was loaded onto the gel, togetherwith 10 μL of SeeBlue plus 2 pre-stained protein standards (Invitrogen,Cat. No. LC5925). The results clearly demonstrated that the asp cloningstrategy described in this Example yield active Asp produced by B.subtilis.

In addition, samples of the same fermentation broths were assayed asfollows: 10 μl of the diluted supernatant was taken and added to 190 μlAAPF substrate solution (conc. 1 mg/ml, in 0.1 M Tris/0.005% TWEEN®, pH8.6). The rate of increase in absorbance at 410 nm due to release ofp-nitroaniline was monitored (25° C.), as it provides a measure of theASP concentration produced. These results indicated that the B. subtilispHPLT-ASP-C1-2 transformants resulted in the production of measurableASP protease.

Example 3 Construction of Combinatorial Mutants

In this Example, the construction of a multiple mutation library of aASP variant is described. The ASP variant that served as the backbonefor the multiple mutation library contains the substitutionsR014I-A064K-T086K-T116E-R123F. This variant was cloned in the pHPLTvector. The QuikChange® multi site-directed mutagenesis (QCMS) kit(Stratagene) was used to construct the library. The 5′ phosphorylatedprimers used to create the library are shown in Table 3-1. It was notedthat HPLC, PAGE or any other type of purified primers gave far betterresults in terms of incorporation of full length primers as well assignificant reduction in primer-containing errors. However, in theseexperiments, purified primers were not used.

TABLE 3-1 Primers and Sequences Primer name Primer sequence ASP-N24E-FWTATCGGATTCGCAGTAGAGGGTGGCTTCATTACTGCCGG (SEQ ID NO: 16) ASP-N24A-FWTATCGGATTCGCAGTAGCCGGTGGCTTCATTACTGCCGG (SEQ ID NO: 17) ASP-N24T-FWTATCGGATTCGCAGTAACAGGTGGCTTCATTACTGCCGG (SEQ ID NO: 18) ASP-N24Q-FWTATCGGATTCGCAGTACAAGGTGGCTTCATTACTGCCGG (SEQ ID NO: 19) ASP-R35E-FWCGGTCACTGCGGAGAGACAGGAGCCACTACTGCC (SEQ ID NO: 20) ASP-R35D-FWCGGTCACTGCGGAGACACAGGAGCCACTACTGCC (SEQ ID NO: 21) ASP-G54D-FWAGGTAGCTCGTTTCCGGACAATGATTATGCATTCGTCCG (SEQ ID NO: 21) ASP-A64K-N67L-FWCCGAACAGGGAAAGGAGTACTGTTGCTTGCCCAAGTCAATAAC (SEQ ID NO: 22)ASP-A64K-N67S-FWCCGAACAGGGAAAGGAGTATCTTTGCTTGCCCAAGTCAATAAC (SEQ ID NO: 23)ASP-A64K-N67A-FWCCGAACAGGGAAAGGAGTAGCTTTGCTTGCCCAAGTCAATAAC (SEQ ID NO: 24)ASP-G78D-T86K-FWTAACTACTCGGGCGACAGAGTCCAAGTAGCAGGACATAAAGCC (SEQ ID NO: 25) ASP-R123F-GTCTTTGGACTTATCCAAACGACGGTTTGTGCCGAACC (SEQ ID NO: 26) R127Q-FWASP-R123F- GTCTTTGGACTTATCAAAACGACGGTTTGTGCCGAACCAG (SEQ ID NO: 27)R127K-FW ASP-R123F-GTCCGAGGACTTATCTACACGACGGTTTGTGCCGAACC (SEQ ID NO: 28) R127Y-FW K159F-FWGGTGGTTCTGGAAATTGTTTCACGGGGGGAACAACATTC (SEQ ID NO: 29) K159E-FWGGTGGTTCTGGAAATTGTGAGACGGGGGGAACAACATTC (SEQ ID NO: 30) K159N-FWGGTGGTTCTGGAAATTGTAACACGGGGGGAACAACATTC (SEQ ID NO: 31) R159K-FWGGTGGTTCTGGAAATTGTAAGACGGGGGGAACAACATTC (SEQ ID NO: 32)pUC18-ASP Preparation

The ASP variant containing the substitutionsR014I-A064K-T086K-T116E-R123F was cloned from the pHPLT vector into thepUC18 vector (Invitrogen, cat. no 15363013; See, FIG. 1), using the PstIand HindIII restriction sites. Subsequently, the pUC18-ASP plasmid (See,FIG. 2) was electroporated to electrocompetent E. coli cells(Invitrogen, cat. no C4040-52, One Shot® TOP10 Electrocomp™ E. coli,dam+) and selective growth on agar plates containing 100 mg/Lampicillin, resulted in pUC18-ASP plasmid harboring E. coli cells. Thismethod was used to ensure methylated ASP DNA at GATC sites, needed toperform the QCMS protocol, because the plasmid pHPLT-ASP-C1-2 does notgrown in E. coli.

Miniprep DNA was prepared from E. coli cells harboring the pUC18-ASPplasmid. Specifically, the strain was grown overnight in 10 mL of 2×TYmedium with 100 ppm of ampicillin, after which the cells were spun down.The Qiagen spin miniprep DNA kit (cat. No. 27106) was used for preparingthe plasmid DNA by the steps outlined in the Qiagen miniprep kit manual.The miniprep DNA was eluted with 50 uL of Qiagen buffer EB provided inthe kit.

Multiple Mutation Library Construction

Sites to be used for multiple mutation library construction were chosenas follows. For each property required in the final molecule, mutationswere evaluated as “Up” or “Down,” with sites defined as productive. At aminimum, sites to be combined should be productive for each propertydesired, and at a minimum, for the most important property. To ensurethat the libraries had the highest probability for improving ormaintaining all important properties, the following criteria were used.

For each property, a cut-off value for ΔΔ G was established. Forexample, for a process taking place at 25° C. ΔΔ G values <0.06 Kcalrepresent those mutations no worse than 90% of the parent proteinsactivity, while ΔΔ G values >1.13 Kcal represent those mutations thatare 125% of the parent proteins activity.

The multiple mutation library was constructed as outlined in theStratagene QCMS kit, with the exception of the primer concentration usedin the reactions. Specifically, 1 μL of the methylated, purifiedpUC18-ASP plasmid (about 70 ng) was mixed with 15 μL of steriledistilled water, 1.5 μL of dNTP, 2.5 μL of 10× buffer, 1 μL of theenzyme blend and 1.0 μL mutant primer mix (for a total of 100 pmol ofprimers). The primer mix was prepared using 10 μL of each of theeighteen mutant primers (100 pmol/μL); adding 50 ng of each primer forthe library as recommended by Stratagene, resulted in fewer mutations ina previous round of mutagenesis. Thus, the protocol was modified in thepresent round of mutagenesis to include a total of 100 pmol of primersin each reaction. The cycling conditions were 95° C. for 1 min, followedby 30 cycles of 95° C. for 1 min, 55° C. for 1 min, and 65° C. for 12min, in an MJ Research PTC-200 thermocycler using thin-walled 0.2 mL PCRtubes. The reaction product was digested with 1 μL of DpnI from the QCMSkit by incubating at 37° C. overnight. An additional 0.5 μL of DpnI wasadded, and the reaction was incubated for 1 hour.

Subsequently, the library DNA (mutagenized single stranded pUC18-ASPproduct) was electroporated to electrocompetent E. coli cells(Invitrogen, cat. no C4040-52, One Shot® TOP10 Electrocomp™ E. coli,dam+) and selective growth on agar plates containing 100 mg/L ampicillinresulted in the ASP multiple mutation library in E. coli cells. Colonies(tens of thousands) were harvested and the Qiagen spin miniprep DNA kit(cat. No. 27106) was used for preparing the plasmid DNA by the stepsoutlined in the Qiagen miniprep kit manual. The miniprep DNA was elutedwith 50 uL of Qiagen buffer EB provided in the kit.

Miniprep DNA was digested using the PstI and HindIII DNA restrictionenzymes. The ASP library fragment mix (PstI×HindIII) was gel purifiedand cloned in the 4154 basepair HindIII×PstI pHPLT vector fragment by aligase reaction using Invitrogen T4 DNA Ligase (Cat. No. 15224-025),utilizing Invitrogen's protocol as recommended for general cloning ofcohesive ends). In another approach, synthetic ASP library fragmentswere produced by GeneArt. These ASP library fragments were also digestedwith PstI and HindIII, purified and cloned in the 4154 basepairHindIII×PstI pHPLT vector fragment by a ligase reaction.

To transform the ligation reaction mix directly into Bacillus cells, thelibrary DNA (ASP library fragment mix cloned in pHPLT) was amplifiedusing the TempliPhi kit (Amersham cat. #25-6400). For this purpose, 1 μLof the ligation reaction mix was mixed with 5 μL of sample buffer fromthe TempliPhi kit and heated for 3 minutes at 95° C. to denature theDNA. The reaction was placed on ice to cool for 2 minutes and then spundown briefly. Next, 5 μL of reaction buffer and 0.2 μL of phi29polymerase from the TempliPhi kit were added, and the reactions wereincubated at 30° C. in an MJ Research PCR machine for 4 hours. The phi29enzyme was heat inactivated in the reactions by incubation at 65° C. for10 min in the PCR machine.

For transformation of the libraries into Bacillus, 0.1 μL of theTempliPhi amplification reaction product was mixed with 500 μL ofcompetent B. subtilis cells (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32,ΔamyE::(xylR,pxylA-comK) followed by vigorous shaking at 37° C. for 1hour and 100 and 500 μL was plated on HI-agar plates containing 20 ppmneomycin sulfate (Sigma, Cat. No. N-1876; contains 732 μg neomycin permg) and 0.5% skim milk. Ninety-five clones from the library were pickedfor sequencing.

The mutagenesis worked well, in that only 14% of the clones were equalto the backbone sequence (ASP with R014I-A064K-T086K-T116E-R123F), about3% of clones had extra mutations. The remaining of the sequenced clones(72%) were all mutants, and of these about 94% were unique mutants. Thesequencing results for the library are provided below in Table 3-3.

TABLE 3-3 Variants of ASP with R014I-A064K-T086K-T116E-R123F G54D N24AN24Q N24T N67S R127K R159F R159K R159K R159N R159N G78D R159F N24Q R35EN67S R159E R127K R159E R127K R159K R127K R159N R127Q R159K R35D R159ER35D R159K R35E R159K G54D R127K R159K G78D R127K R159K G78D R127K R159EG78D R127Q R159K N24A N67A R159K N24A N67S R159K N24E R35D G78D N24TN67S R159E N67L G78D R159K R35D G78D R159K N24A R35E G78D R159N N24DR35D G78D R159F N24E G54D G78D R159K N24E R35D G78D R127K R159N N24QG54D G78D R159N N24Q N67L G78D R159E N24Q R35D R127K R159K N24T R35DG78D R159K N24T R35D G78D R159K N67S G78D R127K R159K R35D G78D R127KR159E R35D G78D R127K R159N R35D G78D R127Q R159K R35E G54D N67A R159FR35E N67S G78D R127Q N24A G54D N67S G78D R159F N24A R35D N67A G78D R159FN24Q R35D N67L G78D R159K N24Q R35D N67L G78D R159N N24Q R35D N67S R127KR159E N24Q R35E N67A R127K R159E N24Q R35E N67A G78D R159E N24T N67AG78D R127Q R159N N24T R35E N67A G78D R127Q R35E G54D N67S G78D R159KN24A G54D N67S G78D R127K R159K N24A R35E N67S G78D R127K R159K N24ER35E G54D N67S R127K R159N N24Q R35D N67S G78D R127K R159F N24T G54DN67S G78D R127Y R159E N24E R35E G54D N67S G78D R127K R159K

Example 4 Preparation of ASP Variant Crude Enzyme Samples

The Asp variant proteins were produced by growing the B. subtilistransformants (described above and in U.S. patent application Ser. No.10/576,331) in 96 well MTP at 37° C. for 68 hours in MBD medium (a MOPSbased defined medium). MBD medium was made essentially as known in theart (See, Neidhardt et al., J. Bacteriol., 119: 736-747 [1974]), exceptthat NH₄Cl₂, FeSO₄, and CaCl₂ were left out of the base medium, 3 mMK₂HPO₄ was used, and the base medium was supplemented with 60 mM urea,75 g/L glucose, and 1% soytone. Also, the micronutrients were made up asa 100× stock containing in one liter, 400 mg FeSO₄.7H₂O, 100 mgMnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100mg NaMoO₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1M CaCl₂, and 10 ml of0.5 M sodium citrate.

In the following Examples, tests conducted on various mutants of ASP aredescribed. The methods described above in Example 1 were used. In thefollowing Tables, “Variant Code” provides the wild-type amino acid, theposition in the amino acid sequence, and the replacement amino acid(i.e., “F001A” indicates that the phenylalanine at position 1 in theamino acid sequence has been replaced by alanine in this particularvariant).

Example 5 Casein Hydrolysis Activity of Multiply-Substituted Variants

In this Example, experiments conducted to determine the caseinolyticactivity of various multiply-substituted ASP variants are described. Inthese experiments, the protocol used is described in Example 1, above.The following table provides the variants, including the mutations ineach variant, as well as the casein activity for those with activitygreater than that of wild-type ASP plus 1 standard deviation (≧1.13activity units).

TABLE 5-1 Caseinolytic Activity Casein Variants Activity R14L-R79T 1.61G12D-R35H-R159E 1.27 G12D-R35H-R123Q 1.30 G12D-R35H-R123F-R159E 1.45G12D-R35H 1.69 G12D-R35E-R159E 1.22 G12D-R35E-R123Q-R159E 1.16G12D-R35E-R123Q 1.14 G12D-R35E 1.43 G12D-R35D-R159E 1.18G12D-R35D-R123Q-R159E 1.27 G12D-R35D 1.64 G12D-R159E 1.49 G12D-R14Q-R35H1.39 G12D-R14I-R35H 1.41 N024E-G049A-A093H--R127K- 1.15A143N-R159K-I181Q N24M-S76V-A93H-R127K- 1.13 R159KR14I-N24E-R35D-A64K-N67A- 1.13 G78D-R123F-R159K-D184T

The following table provides the variants, including the mutations ineach variant, as well as the casein activity for those with activitygreater than that of wild-type ASP plus 1 standard deviation (>0.85activity units).

TABLE 5-2 Caseinolytic Activity Variant Casein Activity R127A-R159K 0.86R14I-G65Q 1.04 R14I-G65Q-R159K 0.99 R14I-S76V 1.43

As indicated by these results, numerous multiply-substituted variantsperformed better than the wild-type ASP in this assay system.

Example 6 Keratin Hydrolysis Activity of Multiply-Substituted Variants

In this Example, experiments conducted to determine the keratinolyticactivity of various multiply-substituted ASP variants are described. Inthese experiments, the protocol used is described in Example 1, above.The following table provides the variants, including the mutations ineach variant, as well as the keratin hydrolysis activity for those withactivity greater than that of wild-type ASP plus 1 standard deviation(≧1.1 performance index).

TABLE 6-1 Keratin Hydrolysis Activity Assay Results Keratin HydrolysisVariants [Perf. Ind.] N024E-G049A-A093H-- 1.32 R127K-A143N-R159K- I181QN024E-G049A-A093H- 1.37 S099A-R127K-A143N- R159K-I181T-V090IN024E-G049A-A093S- 1.28 S099D-R127K-A143N- R159K-I181QN024Q-G049A-A093S- 1.17 S099A-R127K-A143N- R159K-I181QN024Q-G049A-A093S- 1.19 S099A-R127K-A143N- R159K-I181TN024Q-G049A-A093S- 1.13 S099N-R127K-A143N- R159K-I181QN024T-G049A-A093S- 1.12 S099A-R127K-A143N- R159K-I181TN024W-G049A-A093S- 1.13 S099A-R127K-A143N- R159K-I181QN24A-G54E-S76D-A93G- 1.53 R127K-R159K N24E-A93G-R127K- 1.33 R159KN24E-G54L-S76E-A93G- 1.21 R127K-R159K N24E-G54Q-A93S-R127K- 1.37 R159KN24E-S76D-A93T-R127K- 1.64 R159K N24M-G54E-A93H- 1.24 R127K-R159KN24M-G54E-S76N-A93S- 1.24 R127K-R159K N24Q-A93G-R127K- 1.17 R159KN24Q-G54D-S76L-A93G- 1.15 R127K-R159K N24Q-G54I-S76E-A93H- 1.12R127K-R159K N24T-G54D-S76V-A93G- 1.11 R127K-R159K N24T-G54E-S76V-A93H-1.28 R127K-R159K N24W-G54D-A93H- 1.20 R127K-R159K N24W-S76E-A93G- 1.13R127K-R159K R014I-S076A-A093G- 1.27 R127K-R159K-I181T R014I-S076A-A093H-1.22 R127K-R159K-I181Q R014I-S076D-A093H- 1.40 R127K-R159K-I181QR014I-S076D-A093H- 1.46 R127K-R159K-I181T R014I-S076D-A093S- 1.49R127K-R159K-I181T R014I-S076E-A093S- 1.54 R127K-R159K-I181QR014I-S076E-A093T- 1.34 R127K-R159K-I181K R014I-S076I-A093S- 1.27R127K-R159K-I181Q R014I-S076N-A093H- 1.18 R127K-R159K-I181QR014I-S076T-A093G- 1.22 R127K-R159K-I181Q R014I-S076V-A093H- 1.23R127K-R159K-I181Q R014K-S076A-A093S- 1.15 R127K-R159K-I181TR014K-S076E-A093H- 1.14 R127K-R159K-I181T R014K-S076E-A093S- 1.19R127K-R159K-I181T R014K-S076T-A093H- 1.11 R127K-R159K-I181QR014L-S076A-A093H- 1.36 R127K-R159K R014L-S076A-A093H- 1.24R127K-R159K-I181Q R014L-S076D-A093H- 1.32 R127K-R159K-I181TR014L-S076E-A093H- 1.30 R127K-R159K-I181K R014M-S076A-A093G- 1.26R127K-R159K-I181T R014M-S076A-A093H- 1.15 R127K-R159K-I181TR014M-S076A-A093S- 1.22 R127K-R159K-I181K R014M-S076A-A093S- 1.24R127K-R159K-I181T R014M-S076A-A093T- 1.13 R127K-R159K-I181QR014M-S076D-A093S- 1.36 R127K-R159K-I181T R014M-S076E-A093G- 1.25R127K-R159K-I181T R014M-S076E-A093H- 1.42 R127K-R159K-I181TR014M-S076E-A093S- 1.46 R127K-R159K-I181T R014M-S076N-A093G- 1.11R127K-R159K-I181K R014M-S076N-A093G- 1.21 R127K-R159K-I181TR014M-S076N-A093H- 1.23 R127K-R159K-I181T R014M-S076N-A093S- 1.25R127K-R159K-I181T R014M-S076V-A093G- 1.21 R127K-R159K-I181QR14I-N24Q-A64K-G78D- 1.11 R123F-R159K-D184T R14I-N24A-A64K-N67S- 1.34G78D-R123F-R159K- D184T R14I-N24Q-R35D-A64K- 1.21 N67S-R123F-R159K-D184T R14I-N24E-A64K-R123F- 1.31 R127K-R159K-D184T R14I-N24Q-A64K-R123F-1.28 R127Q-R159K-D184T R14I-N24Q-A64K-G78D- 1.21 R123F-R127Q-R159N-D184T R14I-N24E-A64K-N67L- 1.49 G78D-R123F-R159K- D184TR14I-N24Q-R35D-A64K- 1.26 N67S-R123F-R159K- D184T R127Q-R159K 1.14G78D-R127K-R159K 1.24 N67S-G78D-R127K- 1.39 R159K R35D-R159K 1.17G78D-R127Q-R159K 1.26 N24A-N67A-R159K 1.17 T36S-R127Q-R159E 1.77S15E-T121E-R123Q 1.32 S15E-R35H-R159E 1.23 S15E-R35E 1.33 S15E-R159E1.12 S15E-R123Q 1.54 S15E-R123E 1.92 R79T R127Q R179Q 1.55 R35H-R159E1.12 R35H-R127Q-R159E 1.23 R35F R61S R159Q 1.24 R35F R159Q 1.39R35E-R159E 1.52 R35E-R127Q 1.21 R35D-R159E 1.52 R35D-R127Q 1.29 R16QR79T R159Q R179Q 1.36 R16Q R79T R127Q 1.68 R16Q R79T R123L 1.35 R16QR79T 1.24 R16Q R35F R123L R159Q 1.20 R16Q R159Q R179Q 1.14 R16Q R127QR159Q 1.35 R16Q R123L R159Q 1.32 R14Q-T121E 1.44 R14Q-R35E-T121E 1.11R14Q-R35E-R159E 1.20 R14Q-R35E 1.49 R14Q-R35D-R127Q 1.39 R14Q-R35D 1.46R14L R79T R127Q R159Q 1.30 R14L R61S R79T R123L 1.30 R14L R35F R61S 1.27R14L R127Q R159Q 1.47 R179Q R14L R123L R159Q 1.34 R14I-R35E-R127Q 1.74R14I-R35E-R123E 1.19 R14I-R35D-R159E 1.20 R14E-R35H-R127Q 1.60R127Q-R159E 2.44 R127Q R159Q 1.37 R123Q-R159E 1.64 R123Q-R127Q-R159E1.34 R123L R159Q 1.51 R123L R127Q R159Q 1.11 R123F-R159E 1.64R123E-R127Q-R159E 1.15 R123E-R127Q 1.65 G12D-S15E-R35H-R159E 1.40G12D-S15E-R35D 1.59 G12D-S15E-R159E 1.34 G12D-S15E 1.81 G12D-R35H-T121E-1.96 R123Q G12D-R35H-R159E 1.98 G12D-R35H-R127Q- 1.73 R159EG12D-R35H-R123Q 2.43 G12D-R35H-R123F- 1.44 R159E G12D-R35H 2.02G12D-R35E-R159E 1.47 G12D-R35E-R123Q- 1.29 R159E G12D-R35E-R123Q 2.28G12D-R35E 1.98 G12D-R35D-R159E 1.23 G12D-R35D-R127Q 1.59 G12D-R35D 2.23G12D-R159E 2.46 G12D-R14Q-R35H 2.20 G12D-R14Q-R35D-R123H 1.20G12D-R14Q-R159E 1.55 G12D-R14I-R35H 2.82 G12D-R14E 2.00 G12D-R127Q-R159E1.83 G12D-R123E-R159E 1.26

The following table provides the variants, including the mutations ineach variant, as well as the keratinolytic activity for those withactivity greater than that of wild-type ASP plus 1 standard deviation(>1.2 Performance Index).

TABLE 6-2 Kertain Hydrolysis Assay Results Keratin Hydrolysis Variant[Perf. Ind.] R127A-R159K 1.84 R14I-G65Q 1.96 R14I-G65Q-R127A- 1.42 R159KR14I-G65Q-R159K 1.78 R14I-R127A 1.23 R14I-R127A-R159K 1.45 R14I-R159K2.69 R14I-R35F 1.32 R14I-R35F-G65Q 1.50 R14I-R35F-G65Q- 1.66 R127A-R159KR14I-R35F-R159K 1.71 R14I-S76V 2.39 R14I-T36S-G65Q- 1.90 R127A-R159KR35F-R127A-R159K 1.75

As indicated by these results, numerous multiply-substituted variantsperformed better than the wild-type ASP in this assay system.

Example 7 Thermostability of Multiply-Substituted Variants

In this Example, experiments conducted to determine the thermostabilityof various multiply-substituted ASP variants are described. In theseexperiments, the protocol used is described in Example 1, above. Thefollowing table provides the variants, including the mutations in eachvariant, as well as the residual casein activity for those with activitygreater than that of wild-type ASP plus 1 standard deviation (≧48%residual activity).

TABLE 7-1 Thermostability Assay Results Thermostability Variants [% Res.Act] T121E-R123F-R159E 61 R79T-R127Q-R179Q 55 R16Q-R79T-R127Q 55R16Q-R79T-R123L-R159Q- 61 R179Q R16Q-R79T-R123L 65 R16Q-R79T 64R16Q-R123L-R159Q 49 R14Q-T121E 59 R14L-R79T 71 R123L-R159Q 54R123L-R127Q-R159Q 69 G12D-S15E-R35D-R123F- 59 R159E G12D-S15E-R159E 54G12D-S15E 64 G12D-R35H-T121E-R123Q 57 G12D-R35H-R123Q 59G12D-R35H-R123F-R159E 60 G12D-R35H 57 G12D-R35E-R123Q 51 G12D-R35E 52G12D-R159E 53 G12D-R14Q-S15E-R35D 52 G12D-R14Q-R35H 51 G12D-R14Q-R159E49 G12D-R14E 84 G12D-R127Q-R159E 63 G12D-R123E-R159E 58

As indicated by these results, numerous multiply-substituted variantsperformed better than the wild-type ASP in this assay system.

Example 8 LAS Stability of Multiply-Substituted Variants

In this Example, experiments conducted to determine the LAS stability ofvarious multiply-substituted ASP variants are described. In theseexperiments, the protocol used is described in Example 1, above, with atemperature of 25° C. used. The following table provides the variants,including the mutations in each variant, as well as the residual AAPFactivity for those with residual activity greater than that of wild-typeASP plus 1 standard deviation (≧10% residual activity).

TABLE 8-1 LAS Stability Assay Results LAS Stability Variants [%]T121E-R123F-R159E 73 S15E-T121E-R123Q 87 S15E-R35H-R159E 88 S15E-R35E 82S15E-R35D-T121E- 100 R123Q S15E-R35D-R123Q 90 S15E-R35D-R123F- 97 R159ES15E-R35D 81 S15E-R159E 62 S15E-R127Q 67 S15E-R123Q 66 S15E-R123E 81R79T R127Q R179Q 53 R35H-R159E 82 R35H-R127Q-R159E 89 R35H-R123D-R159E82 R35F R61S R159Q 78 R35F R159Q 44 R35E-T121E-R123E 92 R35E-R159E 93R35E-R127Q 84 R35D-R159E 88 R35D-R127Q-R159E 95 R35D-R127Q 89R35D-R123Q-R159E 94 R16Q R79T R159Q 93 R179Q R16Q R79T R127Q 92 R16QR79T R123L 96 R159Q R179Q R16Q R79T R123L 89 R159Q R16Q R79T R123L 74R16Q R79T 38 R16Q R61S R159Q 95 R179Q R16Q R61S R123L 96 R159Q R16Q R35FR61S R159Q 97 R16Q R35F R159Q 82 R16Q R35F R123L 91 R127Q-R159E 40 R127QR159Q 39 R123Q-R159E 42 R123Q-R127Q-R159E 51 R123L R159Q 36 R123L R127QR159Q 56 R123F-R159E 67 R123E-R127Q-R159E 65 R123E-R127Q 49G12D-S15E-R35H-R159E 100 G12D-S15E-R35H- 100 R123F-R127Q-R159EG12D-S15E-R35E-R159E 103 G12D-S15E-R35D-R127Q 99 G12D-S15E-R35D- 101R123F-R159E G12D-S15E-R35D-R123E 100 G12D-S15E-R35D 97 G12D-S15E-R159E96 G12D-S15E 76 G12D-R35H-T121E- 98 R123Q G12D-R35H-R159E 96G12D-R35H-R123Q- 97 R159E G12D-R35H-R123Q 89 G12D-R35H-R123F- 98 R159EG12D-R35H 79 G12D-R35E-R159E 101 G12D-R35E-R123Q- 96 R159EG12D-R35E-R123Q 92 G12D-R35E 97 G12D-R35D-R159E 95 G12D-R35D-R127Q 98G12D-R35D-R123Q- 103 R159E G12D-R35D 94 G12D-R159E 97 S15E-R35E-R159E 93S15E-R35E-R127Q- 102 R159E R159Q R16Q R35F 54 R16Q R159Q R179Q 83 R16QR159Q 69 R16Q R127Q R179Q 88 R16Q R127Q R159Q 76 R16Q R123L R159Q 85R14Q-T121E 88 R14Q-R35E-T121E 96 R14Q-R35E-R159E 91 R14Q-R35E 94R14Q-R35D-R127Q 88 R14Q-R35D-R123E- 93 R159E R14Q-R35D-R123D- 99 R159ER14Q-R35D 85 R14Q-R123Q 87 R14L R79T R127Q 90 R159Q R14L R79T 32 R14LR61S R79T R123L 97 R14L R61S R123L 85 R14L R35F R79T R123L 90 R159Q R14LR35F R61S 80 R14L R127Q R159Q 86 R179Q R14L R123L R159Q 91R14I-R35E-T121E-R159E 100 R14I-R35E-R127Q 97 R14I-R35E-R123E 95R14I-R35D-R159E 98 R14I-R35D-R127Q- 99 R159E R14E-S15E-R35H 92R14E-R35H-R127Q 91 R14D-S15E-R35E-R159E 98 R14D-R35H-R123Q- 98 R159EG12D-R14Q-S15E-R35D 101 G12D-R14Q-R35H 88 G12D-R14Q-R35E- 100R127Q-R159E G12D-R14Q-R35D- 100 R123H G12D-R14Q-R159E 96 G12D-R14I-R35H94 G12D-R14E 85 G12D-R14D-R35H- 100 R123D-R127Q G12D-R127Q-R159E 93G12D-R123E-R159E 99 R127A-R159K 24 R14I-G65Q 13 R14I-G65Q-N67L-R159K 11R14I-G65Q-N67L-Y75G- 11 R127A-R159K R14I-G65Q-R159K 33 R14I-G65Q-S76V-11 R127A-R159K R14I-R127A 71 R14I-R127A-R159K 77 R14I-R159K 58 R14I-R35F64 R14I-R35F-G65Q 47 R14I-R35F-G65Q-R127A- 76 R159KR14I-R35F-N67L-R127A- 61 R159K R14I-R35F-R127A- 81 R159K R14I-R35F-R159K70 R14I-S76V 14 R14I-T36S-G65Q-R127A- 32 R159K R35F-R127A-R159K 57R35F-S76A-R127A 58

In additional experiments, the LAS stability of variants having multiplesubstitutions were tested. In the first set of experiments, the testconditions described in Example 1 were used at a temperature of 25° C.In the following Table, the variants with activity greater thanwild-type, plus one standard deviation (>20% residual activity) arepresented.

TABLE 8-2 Results of LAS Stability Assays at 25° C. LAS-25° C. VariantStability [%] N024A-G049A-A093H- 25 S099N-R127K-A143N- R159K-I181QN024A-S076A-A093H- 47 S099G-R127K-R159K N024A-S076T-A093S- 41S099G-R127K-R159K N024E-G049A-A093G- 31 S099G-R127K-A143N- R159K-I181TN024E-G049A-A093H-- 53 R127K-A143N-R159K- I181Q N024E-G049A-A093H- 75S099A-R127K-A143N- R159K-I181T-V090I N024E-G049A-A093S- 27S099D-R127K-A143N- R159K-I181Q N024H-G049A-A093T- 41 S099A-R127K-A143N-R159K-I181Q N024H-S076A-A093G- 53 S099G-R127K-R159K N024H-S076A-A093H-78 S099G-R127K-R159K N024H-S076A-A093S- 26 S099A-R127K-R159K-G054H-L069H N024H-S076A-A093T- 46 S099G-R127K-R159K N024Q-G049A-A093S-28 S099A-R127K-A143N- R159K-I181T N024Q-S076A-A093H- 58S099A-R127K-R159K- T039N N024Q-S076A-A093H- 38 S099W-R127K-R159KN024Q-S076I-A093T- 25 S099G-R159K N024Q-S076T-A093S- 48 R127K-R159KN024S-S076A-A093G- 46 S099G-R127K-R159K- G054A N024S-S076A-A093H- 41S099T-R127K-R159K N024S-S076A-A093S- 47 S099W-R127K-R159KN024S-S076A-A093T- 21 S099W-R127K N024S-S076T-A093Q- 37S099W-R127K-R159K N024S-S076Y-A093T- 50 S099A-R127K-R159KN024T-G049A-A093G- 48 S099A-R127K-A143N- R159K-I181Q N024T-G049A-A093H-60 S099A-R127K-A143N- R159K-I181Q N024T-G049A-A093H- 58S099A-R127K-A143N- R159K-I181T N024T-G049A-A093H- 21 S099D-R127K-A143N-R159K-I181Q N024T-G049A-A093S- 41 S099A-R127K-A143N- R159K-I181TN24E-G54Q-A93S- 50 R127K-R159K N24E-S76D-A93T- 40 R127K-R159KN24H-A93H-R127K- 41 R159K N24H-G54E-A93G- 46 R127K-R159K N24H-S76D-A93H-35 R127K-R159K N24L-A93G-R127K- 32 R159K N24L-G54E-A93G- 46 R127K-R159KN24L-G54L-A93G- 21 R127K-R159K N24L-G54Q-S76A- 37 A93H-R127K-R159KN24L-S76T-A93G- 24 R127K-R159K N24L-S76T-A93H- 30 R127K-R159KN24M-A93G-R127K- 33 R159K N24M-A93H-R127K- 41 R159K N24M-A93S-R127K- 36R159K N24M-A93T-R127K- 29 R159K N24M-G54E-A93H- 54 R127K-R159KN24M-G54E-S76N- 46 A93S-R127K-R159K N24M-G54I-A93H- 31 R127K-R159K-S187IN24Q-A93G-R127K- 37 R159K N24Q-G54D-A93H- 43 R127K-R159K N24Q-G54I-A93G-25 R127K-R159K R014I-S076D-A093H- 75 R127K-R159K-I181QR014I-S076D-A093S- 41 R127K-R159K-I181T R014I-S076E-A093S- 64R127K-R159K-I181Q R014I-S076E-A093T- 30 R127K-R159K-I181KR014I-S076I-A093S- 23 R127K-R159K-I181Q R014I-S076N-A093H- 68R127K-R159K-I181Q R014I-S076T-A093G- 57 R127K-R159K-I181QR014K-S076A-A093G- 20 R127K-R159K-I181K R014K-S076E-A093H- 20R127K-R159K-I181K R014K-S076T-A093H- 24 R127K-R159K-I181QR014L-S076A-A093H- 69 R127K-R159K R014L-S076A-A093H- 66R127K-R159K-I181Q R014L-S076D-A093H- 39 R127K-R159K-I181TR014L-S076E-A093H- 36 R127K-R159K-I181K R014M-S076A-A093G- 45R127K-R159K-I181K R014M-S076A-A093G- 37 R127K-R159K-I181TR014M-S076A-A093H- 36 R127K-R159K-I181T N024H-S076N-A093Q- 33S099W-R127K-R159K N024H-S076V-A093Q- 29 S099G-R127K-R159KN024L-G049A-A093H- 31 S099A-R127K-A143N- R159K-I181Q N024L-G049A-A093S-23 S099A-R127K-A143N- R159K-I181Q N024L-S076V-A093H- 30 S099G-R127KN024L-S076V-A093S- 29 S099A-R127K-R159K N024M-G049A-A093G- 39S099A-R127K-A143N- R159K-I181Q N024M-G049A-A093H- 26 S099D-R127K-A143N-R159K-I181Q N024M-G049A-A093S- 35 S099A-R127K-A143N- R159K-I181QN024M-G049A-A093S- 23 S099W-R127K-A143N- R159K-I181Q N024Q-G049A-A093H-40 S099A-R127K-A143N- R159K-I181T N024Q-G049A-A093S- 50S099A-R127K-A143N- R159K-I181Q N024T-G049A-A093T- 27 S099A-R127K-A143N-R159K-I181T N024T-S076N-A093Q- 26 S099T-R127K-R159K N024T-S076T-A093T-37 S099N-R127K-R159K N024V-S076A-R127K- 60 R159K N024V-S076V-A093Q- 33S099G-R127K-R159K N024W-A093G-S099W- 33 R127K-R159K N024W-G049A-A093H-35 S099A-R127K-A143N- R159K-I181Q N024W-G049A-A093S- 22S099A-R127K-A143N- R159K-I181K N024W-G049A-A093S- 25 S099A-R127K-A143N-R159K-I181Q N024W-S076A-A093T- 60 S099A-R127K-R159K N024W-S076I-A093Q-26 S099G-R127K-R159K N024W-S076N-A093T- 30 S099G-R159KN024W-S076T-A093H- 44 S099A-R127K-R159K N024W-S076T-A093H- 34S099W-R127K-R159K N024W-S076V-A093H- 26 S099A-R127K-R159KN024W-S099W-R127K- 32 R159K N24A-G54E-S76D- 50 A93G-R127K-R159KN24E-A93G-R127K- 53 R159K N24E-G54L-S76E- 22 A93G-R127K-R159KN24Q-G54I-S76E-A93H- 22 R127K-R159K N24Q-G54Q-A93G- 32 R127K-R159KN24Q-G54Q-S76T- 30 A93H-R127K-R159K N24Q-S76A-A93G- 37 R127K-R159KN24T-G54D-S76V- 26 A93G-R127K-R159K N24T-G54E-S76V- 38 A93H-R127K-R159KN24T-G54I-A93G- 38 R127K-R159K N24T-G54N-A93H- 27 R127K-R159KN24T-G54Q-S76N- 40 A93G-R127K-R159K N24T-G54Q-S76V- 26 R127K-R159KN24T-S76I-R127K- 25 R159K N24T-S76L-A93G- 20 R127K-R159KN24W-A93G-R127K- 35 R159K N24W-G54D-A93H- 35 R127K-R159K N24W-G54I-S76A-26 A93H-R127K-R159K N24W-S76A-A93H- 36 R127K-R159K N24W-S76E-A93G- 21R127K-R159K R014I-S076A-A093G- 44 R127K-R159K-I181T R014I-S076A-A093H-54 R127K-R159K-I181K R014I-S076A-A093H- 71 R127K-R159K-I181QR014I-S076A-A093H- 38 R127K-R159K-I181T R014M-S076A-A093S- 49R127K-R159K-I181K R014M-S076A-A093S- 40 R127K-R159K-I181TR014M-S076A-A093T- 57 R127K-R159K-I181Q R014M-S076D-A093S- 35R127K-R159K-I181T R014M-S076E-A093G- 32 R127K-R159K-I181TR014M-S076E-A093H- 32 R127K-R159K-I181T R014M-S076E-A093S- 39R127K-R159K-I181T R014M-S076N-A093G- 42 R127K-R159K-I181KR014M-S076N-A093G- 36 R127K-R159K-I181T R014M-S076N-A093H- 75R127K-R159K-I181Q R014M-S076N-A093H- 42 R127K-R159K-I181TR014M-S076N-A093S- 37 R127K-R159K-I181T R014M-S076N-A093T- 33R127K-R159K-I181T R014M-S076T-A093H- 37 R127K-R159K-I181KR014M-S076V-A093G- 25 R127K-R159K-I181Q R014M-S076V-A093H- 22R127K-R159K-I181Q

In further experiments, yet other test conditions were used. In one setof experiments, the LAS stability was tested at a higher temperature(35° C.), using the protocol described in Example 1. In the followingtable, the percent residual activity for variants that exhibitedactivity greater than wild-type plus one standard deviation (>5%residual activity) is presented.

TABLE 8-3 Results of LAS Stability Assay at 35° C. LAS Stability (35°C.) Variant [%] G54E-R14L 32 G54L-R127S 6 N24D-G54F- 6 R127C N24E-R127S7 N24E-R159C 7 N24G-G54I-R127S 10 N24H-R159Y-T46I 52 N24I-R127V-R14V 18N24T-R127Q- 11 R179F R127A-R159V- 12 R179F R127C-R14W 7 R127S-R159N- 50R123L R14A-N24F- 25 R159L R14A-R127L 28 R14A-R127Y- 28 R159W R14A-R159W9 R14C-S114F- 25 R159G R14F-R127L- 19 R159F R14F-R127Q- 37 R159WR14F-R127S- 32 R159V R14F-R127V- 16 R159F R14G-N24L- 13 R159G R14G-N24S-47 R127C R14G-R127C- 58 G63E R14G-R127G 19 R14T-N24T- 42 R127Y-R159WR14T-R127Y 29 R14V-N24A- 26 R127I-R159A R14V-N24D- 69 R127C R14V-N24G- 7P189S R14V-N24S 9 R14V-N24Y- 37 R127S-R159G R14V-R127A 48 R14V-R127C- 62R159S R14V-R127M- 63 R159V R14G-R127P 9 R14L-N24S-R159F 20 R14L-N24V- 31R127S-R159I R14L-R123L 25 R14L-R127C- 32 R159G R14L-R127S 22 R14L-R127S-31 R159G R14L-R127V 17 R14L-R127V- 27 R159F R14L-R127W- 47 R123YR14L-R127Y 10 R14L-R127Y- 30 R159F R14L-R159G 23 R14L-R159L 30R14L-R159S 43 R14L-R159V 40 R14L-R159W 5 R14M-N24L- 39 R159S-R123VR14M-R159F 17 R14Q-R123F 56 R14S-N24E- 13 R127W R14S-N24L- 17 R159GR14S-R127L- 41 R159F R14S-R127V 33 R14T-R14P-R159F 33 R14T-N24A 6R14T-N24T- 69 R127Q R14V-R127S- 47 R159G R14V-R127T- 49 R159Y R14V-R127V27 R14V-R159F 33 R14V-R159V 53 R14V-R159W 12 R14W-N24T- 48 R123ER14W-R123L 6 R14W-R123V 19 R14W-R127Q- 13 R159W R14W-R159V 13 R159V-G49D6

In additional experiments, variants were tested at 35° C., using theprotocol described in Example 1. In the following Table, results forvariants with G12D and R35E, and >70% residual activity are presented.

TABLE 8-4 Results of LAS Stability Assay at 35° C. LAS-35° C. G12D-R35EVariant Stability [%] G012D-R035E-G065E 73 G012D-R035E-G065E 73G012D-R035E-Q081P 79 G012D-R035E-R016S- 77 A064T G012D-R035E-R159W 74G012D-R035E-R179I 82 G012D-R035E-S092T- 71 I181V

In additional experiments, yet other conditions were used. In one set ofexperiments, the performance indices for numerous multiple substitutedvariants were determined using the protocol in Example 1 and tested at25° C. In the following table, the performance index results (PI) forvariants with residual activity greater than wild-type plus 1 standarddeviation (>1.1 performance index) are presented.

TABLE 8-5 LAS Assay Stability Results LAS (25° C.) Variant [Perf. Ind.]R14I-N24A-A64K-R123F- 2.97 R159E-D184T R14I-A64K-R123F-R159F- 3.99 D184TR14I-A64K-R123F-R159E- 3.82 D184T R14I-N24Q-R35E-A64K- 3.89 R123F-D184TR14I-N24Q-A64K-N67S- 1.24 R123F-R159F-D184T R14I-N24A-R35E-A64K- 1.20N67S-R123F-R159E- D184T R14I-N24A-R35E-A64K- 1.20 N67S-G78D-R123F-D184TR14I-N24A-R35D-A64K- 1.72 G78D-R123F-R127K- R159E-D184TR14I-N24A-R35D-A64K- 2.88 R123F-R127K-R159F- D184T R14I-N24T-R35D-A64K-3.75 G78D-R123F-R127Q- R159F-D184T R14I-A64K-R123F-D184T 3.99R14I-N24A-A64K-R123F- 2.94 R159N-D184T R14I-N24Q-R35D-A64K- 1.31N67S-R123F-R159K- D184T R14I-N24E-R35E-A64K- 9.51 G78D-R123F-R127Q-R159F-D184T R14I-N24E-A64K-R123F- 6.64 R127K-R159K-D184TR14I-N24A-A64K-R123F- 2.80 D184T R14I-A64K-G78D-R123F- 1.60R127Q-R159N-D184T R14I-N24A-R35E-A64K- 1.74 G78D-R123F-R159N- D184TR14I-A64K-R123F- 4.27 R127K-R159E-D184T R14I-N24A-R35E-A64K- 1.20N67S-G78D-R123F- R127K-R159F-D184T R14I-A64K-G78D-R123F- 1.71R159E-D184T R14I-N24E-R35D-A64K- 4.23 N67A-G78D-R123F- R159K-D184TN24T-R35D-G78D-R159K 6.53 N24T-R35E-N67A-G78D- 1.36 R127Q N24Q-R35E 6.60R127K-R159N 6.02 R35D-R159E 7.25 R35E-G54D-N67S-G78D- 1.58 R159KN24Q-G54D-G78D-R159N 2.84 R127K-R159E 6.58 R127Q-R159K 5.76N24E-R35E-G54D-N67S- 9.73 R127K-R159N R35D-G78D-R159K 5.17 N67S-R159E2.05 G54D-R127K-R159K 6.11 G78D-R127K-R159K 2.76 G78D-R127K-R159E 2.63R14I-A64K-R123F- 4.25 R127K-R159F-D184T R14I-A64K-R123F-R159E- 4.22D184T R14I-A64K-R123F- 3.93 R159N-D184T R14I-A64K-R123F- 3.83R159K-D184T R14I-A64K-R123F- 4.32 R127Y-R159E-D184T R14I-N24A-R35E-A64K-2.40 N67A-G78D-R123F- D184T R14I-A64K-R123F- 4.08 R127Y-R159K-D184TR14I-N24Q-A64K-R123F- 3.92 R127Q-R159K-D184T R14I-A64K-R123F- 4.29R159K-D184T R14I-N24Q-A64K-G78D- 1.87 R123F-R127Q-R159N- D184TR14I-N24E-A64K-N67L- 1.33 G78D-R123F-R159K- D184T R14I-N24A-R35E-A64K-1.91 G78D-R123F-R127K- R159E-D184T R14I-N24Q-R35D-A64K- 1.33N67S-R123F-R159K- D184T R14I-N24A-R35D-A64K- 1.22 N67A-R123F-R159F-D184T R14I-N24E-R35E-G54D- 2.64 A64K-N67L-G78D-R123F- R127K-D184TN24E-R35D-G78D- 11.02 R127K-R159N R35D-G78D-R127K- 5.38 R159NN24A-R35E-G78D-R159N 4.03 N24Q-R35D-N67S- 2.13 R127K-R159EN24T-R35D-G78D-R159K 6.73 N67S-G78D-R127K- 1.12 R159K N24Q-R35D-R127K-7.33 R159K N24E-G54D-G78D-R159K 8.72 R35D-R159K 6.89 R35E-R159K 7.66R127K-R159K 5.82 R35E-N67S-G78D-R127Q 2.67 N24E-R35D-G78D 11.21R35D-G78D-R127K- 5.44 R159E N24E-R35E-G54D-N67S- 7.87 G78D-R127K-R159KN24T-N67S-R159E 3.10 N24D-R35D-G78D-R159F 9.11 N24Q-R35D-N67S-G78D- 1.50R127K-R159F R35D-G78D-R127Q- 4.41 R159K G78D-R159F 2.48 N24A-N67S-R159K1.14 G78D-R127Q-R159K 2.63 N24T-G54D-N67S-G78D- 2.42 R127Y-R159E

In additional experiments, the LAS stability of numerous variants wastested at 25° C., using the protocol set forth in Example 1. As above,the selection criterion was residual activity greater than WT+1 standarddeviation (>1.1 Performance Index).

TABLE 8-6 LAS Stability Assay Results LAS Stab-25° Variant [Perf. Ind]R14I-A63K-G78D- 2.66 R123F-D184T R14I-A63K-R123F- 3.53 R159E-D184TR14I-A63K-R123F- 3.48 R159F-D184T R14I-A63K-R123F- 3.33 R159K-D184TR14I-A63K-R123F- 3.83 R159N-D184T R14I-A63K-R123K- 3.38 D184TR14I-A63K-R123Q- 3.58 D184T R14I-A63K-R123Y- 3.62 D184T R14I-A64K-G78D-1.47 T86K-T116E-R123F R14I-A64K-T86K- 5.21 T116E-R123F-R159ER14I-A64K-T86K- 5.11 T116E-R123F-R159K R14I-A64K-T86K- 4.21 T116E-R123KR14I-A64K-T86K- 4.74 T116E-R123Q R14I-A64K-T86K- 4.34 T116E-R123YR14I-G54D-A63K- 2.74 R123F-D184T R14I-G54D-A64K- 4.12 T86K-T116E-R123FR14I-G54D-S76N- 8.53 A93H-R127K-R159K- I181Q R14I-G54D-S76V- 3.53A93S-R127K-R159K- I181K R14I-N24A-A63K- 2.59 R123F-D184T R14I-N24A-A64K-3.11 R14I-S76N-A93H- 8.54 R127K-R159E-I181Q R14I-S76N-A93H- 7.56R127K-R159F-I181Q R14I-S76N-A93H- 9.01 R127K-R159N-I181Q R14I-S76N-A93H-9.11 R127Q-R159K-I181Q R14I-S76N-A93H- 6.88 R127Y-R159K-I181QR14I-S76N-G78D- 5.55 A93H-R127K-R159K- I181Q R14I-S76V-A93S- 1.82R127K-R159F-I181K R14I-S76V-A93S- 2.00 R127K-R159N-I181K R14I-S76V-A93S-2.59 R127Q-R159K-I181K R14I-S76V-A93S- 1.63 R127Y-R159K-I181KR14M-G54D-S76N- 7.52 A93G-R127K-R159K- I181K R14M-N24A-S76N- 1.38A93G-R127K-R159K- I181K R14M-N24E-S76N- 7.17 A93G-R127K-R159K-T86K-T116E-R123F R14I-N24E-A63K- 7.31 R123F-D184T R14I-N24E-A64K- 9.39T86K-T116E-R123F R14I-N24Q-A63K- 4.13 R123F-D184T R14I-N24Q-A64K- 4.51T86K-T116E-R123F R14I-N24T-A63K- 4.35 R123F-D184T R14I-N24T-A64K- 6.54T86K-T116E-R123F R14I-N24TS76N-A93H- 7.43 R127K-R159K-I181QR14I-N24TS76V-A93S- 1.78 R127K-R159K-I181K R14I-N67AS76N- 4.59A93H-R127K-R159K- I181Q R14I-N67LS76N-A93H- 2.71 R127K-R159K-I181QR14I-N67SS76N-A93H- 4.32 R127K-R159K-I181Q R14I-R35D-A64K- 5.72T86K-T116E-R123F R14I-R35D-S76N- 9.45 A93H-R127K-R159K- I181QR14I-R35E-A63K- 3.50 R123F-D184T R14I-R35E-A64K- 6.08 T86K-T116E-R123FR14I-R35E-S76N- 9.00 A93H-R127K-R159K- I181Q R14I-R35E-S76V- 5.22A93S-R127K-R159K- I181K R14I-R35K-A63K- 3.10 R123F-D184T I181KR14M-N24Q-S76N- 3.58 A93G-R127K-R159K- I181K R14M-N24T-S76N- 5.57A93G-R127K-R159K- I181K R14M-N67S-S76N- 1.53 A93G-R127K-R159K- I181KR14M-R35D-S76N- 8.65 A93G-R127K-R159K- I181K R14M-R35E-S76N- 9.25A93G-R127K-R159K- I181K R14M-S76N-A93G- 6.89 R127K-R159E-I181KR14M-S76N-A93G- 5.14 R127K-R159F-I181K R14M-S76N-A93G- 6.71R127K-R159N-I181K R14M-S76N-A93G- 5.85 R127Q-R159K-I181K R14M-S76N-A93G-4.75 R127Y-R159K-I181K R14M-S76N-G78D- 2.13 A93G-R127K-R159K- I181K

As indicated by these results, numerous multiply-substituted variantsperformed better than the wild-type ASP in this assay system.

Example 9 Stain Removal Performance of Multiply-Substituted Mutants

In these experiments, the protocol used is described in Example 1,above. The following table provides the variants, including themutations in each variant for those with activity greater than wild-typeplus one standard deviation (>1.1 Performance Index [PI]). As indicatedby these results, numerous multiply-substituted variants performedbetter than the wild-type ASP in this assay system.

TABLE 9-1 Wash Performance Results BMI LVJ1 Detergent Variant [Perf.Ind] N24F-R159G 1.24 N24F-R159L-R123V 1.22 N24I-R127S 1.15 N24L-R159S1.14 N24S-R159A 1.41 N24V-R159L 1.34 N24Y-R159F 1.14 R14A-N24K-R127S1.32 R14A-R159W 1.12 R14L-N24Y 1.32 R14L-R159G 1.22 R14L-R159S 1.11R14L-T109M 1.14 R14L-T39P 1.26 R14M-R159W 1.18 R14S-N24V 1.34 R14S-N24Y1.32 R14T-N24A 1.16 R14V-N24G-P189S 1.23 R14V-R159W 1.17 R127A-R159K1.23 R14I-G65Q 1.32 R14I-S76V 1.27 R14I-G65Q-N67L- 1.11 R159KR14I-G65Q-R159K 1.32 R14I-S76V 1.48

In additional experiments using liquid detergent, the following resultswere obtained. The variants indicated in the following table exhibitedactivity greater than wild-type plus one standard deviation (>1.1Performance Index), as shown in the following Tables.

TABLE 9-2 Wash Performance Results for LVJ-1 BMI LVJ-1 DetergentVariants [Perf. Ind.] R35F R159Q 1.13 R16Q R79T 1.31 R16Q R35F 1.10 R16QR159Q 1.17 R14L R79T 1.45 R123L R159Q 1.16 G12D-S15E 1.23 G12D-R35H 1.46G12D-R35D 1.13

TABLE 9-3 Wash Performance Results for TIDE ® 2005 BMI Tide2005Detergent Variant [Perf. Ind] N024E-G049A- 1.23 A093H--R127K-A143N-R159K-I181Q N024H-S076A- 1.11 A093S-S099A- R127K-R159K-G054H-L069H N024H-S076A- 1.16 A093T-S099G- R127K-R159K N024L-S076V- 1.11A093S-S099A- R127K-R159K N024Q-G049A- 1.13 A093S-S099A- R127K-A143N-R159K-I181T N024T-G049A- 1.13 A093S-S099A- R127K-A143N- R159K-I181TN24E-G54Q-A93S- 1.17 R127K-R159K N24H-A93H-R127K- 1.14 R159KN24W-S76Y-A93G- 1.22 R127K-R159K R014I-S076A-A093G- 1.22R127K-R159K-I181T R014I-S076A-A093H- 1.23 R127K-R159K-I181KR014I-S076A-A093H- 1.14 R127K-R159K-I181Q R014I-S076A-A093H- 1.15R127K-R159K-I181T R014I-S076D-A093S- 1.10 R127K-R159K-I181TR014I-S076E-A093T- 1.28 R127K-R159K-I181K R014I-S076N-A093H- 1.26R127K-R159K-I181Q R014I-S076V-A093H- 1.22 R127K-R159K-I181QR014I-S076V-A093S- 1.27 R127K-R159K-I181K R014K-S076A- 1.21 A093S-R127K-R159K-I181T R014K-S076E- 1.19 A093H-R127K- R159K-I181KR014K-S076I-A093S- 1.14 R127K-R159K-I181T R014K-S076T- 1.11 A093H-R127K-R159K-I181K R014K-S076T- 1.23 A093H-R127K- R159K-I181T R014K-S076V- 1.11A093H-R127K- R159K-I181K R014L-S076A- 1.34 A093H-R127K- R159KR014M-S076N- 1.28 A093S-R127K- R159K-I181T R014M-S076T- 1.28A093H-R127K- R159K-I181K R014M-S076V- 1.17 A093G-R127K- R159K-I181QR014M-S076W- 1.14 A093H-R127K- R159K-I181K N24H-G54L-S76V- 1.28A93H-R127K-R159K N24L-G54Q-S76A- 1.13 A93H-R127K-R159K N24M-A93G-R127K-1.10 R159K N24M-G54E-A93H- 1.15 R127K-R159K N24M-S76V-A93H- 1.29R127K-R159K N24Q-G54Q-S76T- 1.11 A93H-R127K-R159K N24Q-S76A-A93G- 1.13R127K-R159K N24T-G54Q-S76N- 1.14 A93G-R127K-R159K N24T-S76I-R127K- 1.12R159K N24W-G54D-A93H- 1.20 R127K-R159K N24W-S76A-A93H- 1.14 R127K-R159KN24W-S76T-A93G- 1.12 R127K-R159K N24W-S76V-A93G- 1.12 R127K-R159KR014L-S076A- 1.13 A093H-R127K- R159K-I181Q R014L-S076D- 1.17A093H-R127K- R159K-I181T R014L-S076E- 1.23 A093H-R127K- R159K-I181KR014M-S076A- 1.25 A093G-R127K- R159K-I181K R014M-S076A- 1.12A093G-R127K- R159K-I181T R014M-S076A- 1.18 A093H-R127K- R159K-I181TR014M-S076A- 1.46 A093S-R127K- R159K-I181K R014M-S076A- 1.17A093S-R127K- R159K-I181T R014M-S076A- 1.11 A093T-R127K- R159K-I181QR014M-S076I- 1.30 A093H-R127K- R159K-I181T R014M-S076I-A093S- 1.23R127K-R159K-I181T R014M-S076N- 1.23 A093G-R127K- R159K-I181KR014M-S076N- 1.24 A093G-R127K- R159K-I181T R014M-S076N- 1.39A093H-R127K- R159K-I181T R014M-S076Y- 1.29 A093H-R127K- R159K-I181KR014M-S076Y- 1.14 A093H-R127K- R159K-I181T G012D-R035E- 1.13 D184NG012D-R035E- 1.24 N067K

In further experiments, the following results were obtained. In theseexperiments, the variants were compared to 0.5 ppm wild-type ASP as areference. The results in the following table are provided for variantsthat had activities greater than wild-type plus one standard deviation(>1.1 Performance Index).

TABLE 9-4 Wash Performance Results for TIDE ® SNOW A Detergent BMITIDE-SNOWA Variant Detergent [Perf. ind] R14I-A64K-R123F- 1.27R159F-D184T R14I-A64K-R123F- 1.45 R159F-D184T R14I-N24Q-A64K- 1.38G78D-R123F- R159K-D184T R14I-N24Q-A64K- 1.34 N67S-R123F- R159F-D184TR14I-N24Q-A64K- 1.40 N67A-R123F- R159K-D184T R14I-N24A-A64K- 1.31R159K-D184T R14I-N24E-A64K- 1.23 R123F-R127K- R159K-D184TR14I-N24A-A64K- 1.65 R123F-D184T R14I-A64K-R123F- 1.48 R127K-R159F-D184T R14I-A64K-R123F- 1.38 R159N-D184T R14I-A64K-R123F- 1.66R159K-D184T R14I-A64K-R123F- 1.11 R127Y-R159K- D184T R14I-N24Q-A64K-1.25 R123F-R127Q- R159K-D184T N67S-G78D- R123F-R159K- D184TR14I-A64K-R123F- 1.45 D184T R14I-N24A-A64K- 1.47 R123F-R159N- D184TR14I-N24Q-R35D- 1.23 A64K-N67S- R123F-R159K- D184T R14I-N24Q-A64K- 1.45N67A-R123F- R14I-A64K-R123F- 1.55 R159K-D184T R14I-N24Q-R35D- 1.22A64K-N67S- R123F-R159K- D184T R14I-A64K-N67S- 1.48 G78D-R123F-R127K-R159K- D184T R14I-N24Q-A64K- 1.67 N67A-R123F- R127K-R159K- D184TR127K-R159K 1.53 N24A-N67S-R159K 1.55 N24A-N67A- 1.58 R159K

In additional experiments, the following results were obtained. Thevariants indicated in the following table exhibited activity greaterthan wild-type plus one standard deviation (>1.1 Performance Index).

TABLE 9-5 Wash Performance Results for TIDE ®-SNOW BMI TIDE-SNOWDetergent [Perf. Variant Ind] R14I-A63K-G78D-R123F- 1.39 D184TR14I-A63K-N67A-R123F- 1.50 D184T R14I-A63K-N67L-R123F- 1.23 D184TR14I-A63K-N67S-R123F- 1.48 R14I-A63K-R123K-D184T 1.40R14I-A63K-R123Q-D184T 1.44 R14I-A63K-R123Y-D184T 1.25R14I-A64K-G78D-T86K- 1.26 T116E-R123F R14I-A64K-N67A-T86K- 1.66T116E-R123F R14I-A64K-N67L-T86K- 1.19 T116E-R123F R14I-A64K-T86K-T116E-1.54 R123F-R159K R14I-A64K-T86K-T116E- 1.70 R123K R14I-G54D-A63K-R123F-1.13 D184T R14I-G54D-A64K-T86K- 1.11 T116E-R123F R14I-N24A-A63K-R123F-1.47 D184T R14I-N24A-A64K-T86K- 1.56 T116E-R123F R14I-N24A-S76V-A93S-1.15 R127K-R159K-I181K R14I-N24E-A63K-R123F- 1.36 D184TR14I-N24E-A64K-T86K- 1.26 T116E-R123F R14I-N24Q-A63K-R123F- 1.47 D184TR14I-N24Q-A64K-T86K- 1.57 D184T R14I-A63K-R123F-R159F- 1.75 D184TR14I-A63K-R123F-R159K- 1.70 D184T R14I-A63K-R123F-R159N- 1.67 D184TT116E-R123F R14I-N24Q-S76V-A93S- 1.43 R127K-R159K-I181KR14I-N24T-A63K-R123F- 1.57 D184T R14I-N24T-A64K-T86K- 1.38 T116E-R123FR14I-N24TS76V-A93S- 1.13 R127K-R159K-I181K R14I-N67SS76N-A93H- 1.17R127K-R159K-I181Q R14I-R35E-A63K-R123F- 1.55 D184T R14I-R35K-A63K-R123F-1.20 D184T R14I-S76V-A93S-R127K- 1.12 R159F-I181K R14I-S76V-A93S-R127K-1.31 R159N-I181K R14I-S76V-A93S-R127Y- 1.30 R159K-I181KR14M-N24T-S76N-A93G- 1.11 R127K-R159K-I181K R14M-N67S-S76N-A93G- 1.22R127K-R159K-I181K R14M-S76N-A93G-R127K- 1.41 R159F-I181KR14M-S76N-G78D-A93G- 1.20 R127K-R159K-I181K

Example 10 Stain Removal Performance of Multiply-Substituted Mutants

In this Example, experiments conducted to determine the stain removalability of various multiply-substituted ASP variants are described. Inthese experiments, the following protocols and materials were used.

First, 105 ppm water (˜6 gpg) was prepared by first preparing a 10,000ppm stock solution (3/1 Ca⁺²/Mg⁺²) by dissolving 11.020 g/L calciumchloride dehydrate (CaCl₂.2H₂O) MW 147.01 g/mol and 5.08 g/L magnesiumchloride hexahydrate (MgCl₂.6H₂O) MW 203.3 g/mol in 1000 ml deionizedwater. The 105 ppm solution was prepared by diluting 10.50 ml of the10,000-ppm stock with 989.50 ml deionized water.

A TIDE® 2005 detergent solution (1.6 gm/L) was prepared in the 105 ppmwater by dissolving 11.2 gm TIDE®-2005 in 7 liters of 105 ppm water.

Blood-Milk-Ink (BMI) soiled swatches EMPA 116 (11×8 cm), EMPA117 (11×8cm) both from EMPA Testmaterialien AG and grass soiled swatches EMPA164(10×7.5 cm) from EMPA Testmaterialien AG and Equest grass medium soiled(10×7.5 cm) from Warwick Equest Limited used in these experiments werenumbered using a waterproof pen (on the soiled sides). The soiled sidesof the swatches were measured using Tristimulus Minolta Meter CR-300(Minolta), and analyzed using the equation L (L*a*b), D65 Std.Illuminate, on a white background. Three readings were made per swatch.As grass stains are very sensitive to light, grass-soiled swatches werecovered and stored in the dark until they were used. Every washperformance test was conducted in duplicate.

During the test, extra ballast (more mechanical action) was used. Thisballast comprised 2 pieces of 10×10 cm EMPA 221 (unsoiled cotton, EMPATestmaterialien AG placed in one beaker. These EMPA 221 swatches werenot numbered or measured.

The laundry wash performance test was conducted using a 6 pot, benchmodel Tergotometer (TOM) Model 7243S (United States Testing). Thetemperature was adjusted 15° C. or 60° F. Half of the inside of the TOMwas filled with ice. The wash time was set for 12 minutes. Test beakerswere filled with 1 liter of the detergent solution (made in 105 ppmwater, as described above). Once the solutions have reached 15° C. or60° F., the TOM was started with an agitation of 100 rpm. The variantsamples were then added to the mixture as indicated below. As a control,the TIDE® 2005 detergent solution (Procter & Gamble) without any addedenzyme sample was used. The variants were tested at two differentconcentrations (i.e., at 0.55 ppm and at 2.75 ppm).

Six swatches (with the same soils) were added to each beaker one at atime. In addition, 2 EMPA 221 swatches were added. For example: for BMIsoiled swatches 3 EMPA 116 and 3 EMPA117+2 EMPA 221 (total weight isapproximately 13.25 gram), or, for grass soiled swatches 3 EMPA 164 and3 Equest grass medium soiled+2 EMPA 221 (total weight is approximately13.22 gram). The wash time was then reset on 12 minutes. After 12minutes of washing, all of the EMPA 116 and EMPA 117 swatches wererinsed in a bucket and all the grass swatches were rinsed a separatebucket for 3 minutes under cold running tap water. The EMPA 221 swatcheswere then discarded. The swatches were placed in a spin-dryer for 2minutes (the EMPA 116 and the EMPA 117 swatches were dried separatelyfrom the grass stained-swatches). Subsequently, the grass-stainedswatches were dried in the air, without light or ironing., whereas theEMPA 116 and EMPA 117 swatches were dried by ironing.

After drying, the soiled sides of the swatches were evaluated using theTristimulus Minolta Meter CR-300 with equation L (L*a*b), D65 Std.Illuminate, on a white background, 3 readings per swatch. The percentsoil removal was calculated using the equation:% Soil Removal=(L value after washing−L value before washing)/(L_(0 white cotton) −L value before washing)×100%

In order to obtain a measure for the stain removal of each individualvariant the delta percentage of soil removal was then calculated bysubtracting the percentage of soil removal obtained by the TIDE® 2005detergent solution without any variant from the percentage of soilremoval in the presence of each variant (delta % SR). The results areshown in Table 10.1

In additional experiments, the stain removal abilities of variousmultiply-substituted ASP variants were tested. The test conditions asdescribed above were used with the following modifications. In thesetests a TIDE®2005 SNOW detergent solution (1.5 gm/L) was prepared in the105 ppm water by, dissolving 37.5 gm TIDE®2005 SNOW in 25 liters of 105ppm water. Furthermore, the EMPA 116 BMI soiled swatches were cut intopieces of 10×7.5 cm. The EMPA 117 swatches were replaced by EMPA 116Fixed swatches (CFT) and also cut into pieces of 10×7.5 cm. No EMPA 164swatches were used in these experiments. The variants were tested at oneconcentration of 0.55 ppm. Six swatches (i.e. 6 EMPA 116, or 6 EMPA 116fixed or 6 Equest) were added to each beaker one at a time. In addition,2 EMPA 221 swatches were added. As a control, TIDE® 2005 SNOW detergentsolution (Procter & Gamble), without any added enzyme sample was used.Every wash performance test was conducted in

TABLE 10.1 EMPA EMPA Equest EMPA 116 117 Grass 164 Δ % S.R. Δ % S.R. Δ %S.R. Δ % S.R. Variants/Dosage (n = 6) (n = 6) (n = 6) (n = 6) ASP/0.55ppm 5.2 ± 1.2 4.9 ± 2.4 −4.3 ± 4.3   1.5 ± 1.1 ASP/2.75 ppm 9.7 ± 1.410.7 ± 2.8  −1.8 ± 5.4   3.1 ± 1.6 R123L-R127Q- 6.0 ± 1.5 8.5 ± 2.1 1.7± 3.7 2.1 ± 1.2 R179Q/0.55 ppm R123L-R127Q- 11.3 ± 1.5  14.0 ± 2.7  1.1± 5.0 5.4 ± 1.4 R179Q/2.75 ppm G12D-R35H/ 7.0 ± 1.1 7.9 ± 1.9 −2.2 ±5.0   2.5 ± 1.3 0.55 ppm G12D-R35H/ 10.1 ± 1.7  14.4 ± 1.9  3.2 ± 3.84.5 ± 1.0 2.75 ppm G12D-R35E/ 8.2 ± 1.2 10.0 ± 2.1  1.3 ± 5.4 2.9 ± 1.40.55 ppm G12D-R35E/ 10.6 ± 1.4  16.6 ± 2.1  4.3 ± 4.2 5.4 ± 1.4 2.75 ppmR35E-R123L- 6.1 ± 1.2 6.8 ± 2.1 0.4 ± 4.1 1.0 ± 1.4 R127Q-R179Q/ 0.55ppm R35E-R123L- 8.8 ± 2.1 12.7 ± 1.8  4.7 ± 4.3 1.7 ± 1.4 R127Q-R179Q/2.75 ppmquadruplicate. The results (average delta % SR, Standard deviation (STD)and number of replicates (n)) are shown in FIG. 6.

In another set of experiments, the stain removal ability of numerousmultiply-substituted ASP variants, were tested. The test conditions asdescribed above were used with the following modifications. In thesetests, a TIDE®2005 SNOW detergent solution (1.5 gm/L) containing HEPESbuffer was prepared in the 105 ppm water by dissolving 37.5 gm TIDE®2005 SNOW in 25 liters of 105 ppm water. Then, 30 g Hepes (=5 mM;C₈H₁₈N₂O₄S), was added, the solution was stirred, and the pH adjusted to7.8 with ±19 ml 4N NaOH. It was noted that this solution can be storedfor 36 hours at room temperature. In these tests, only EMPA 116 BMIsoiled swatches of 10×7.5 cm and Equest grass medium soiled swatches of10×7.5 cm were used. The variants were tested at one concentration of0.55 ppm. Six swatches (either 6 EMPA 116, or 6 Equest grass mediumsoiled) were added to each beaker one at a time. In addition, 2 EMPA 221swatches were added. As a control, TIDE® 2005 SNOW detergent solution(Procter & Gamble) with HEPES buffer, without any added enzyme samplewas used. Every wash performance test was conducted in quadruplicate.The results (average □% SR, Standard deviation (STD) and number ofreplicates (n)) are shown in FIG. 7.

As indicated by these results, numerous multiply-substituted variantsperformed better than the wild-type ASP in this assay system.

Example 11 Stain Removal Performance of Multiply-Substituted MutantsTested at Low pH

In this Example, experiments conducted to determine the stain removalperformance of various multiply-substituted ASP variants are described.In these experiments, the protocol used is described in Example 1,above. The following table provides the variants, including themutations in each variant for those with activity greater than that ofwild-type ASP plus 1 standard deviation (1.1 Performance Index).

TABLE 11-1 Stain Removal Performance at Low pH BMI BMI Low pH Low pHVariant [Perf ind] Variant [Perf ind] G54E-R14L 1.40 R127M-R159V 1.70N24D-R127Y- 1.61 R127S-R159G 1.21 R159V R127S-R159L 1.27 N24E-R127S 1.43R127T-R159F 1.28 N24E-R159C 1.14 R127V-R159G 1.36 N24F-R159G 1.42R127Y-R159L 2.14 N24F-R159G-G54E 1.98 R14A-N24K-R127S 1.41N24F-R159L-R123V 1.61 R14A-R127Y- 1.17 N24G-R127Y 1.34 R159WN24H-R159Y-T46I 1.34 R14G-N24S-R127C 1.10 N24I-R127S 1.21 R14G-R127G1.17 N24I-R127V-R14V 1.12 R14L-N24D 1.73 N24L-R159S 1.37 R14L-N24Y 1.19N24S-R159A 2.48 R14L-R123L 2.11 N24V-R127M- 1.33 R14L-R127S 1.47 R159VR14L-R127V 1.18 N24V-R127S-R159H 1.60 R14L-R127Y 1.78 N24V-R159L 1.41R14L-R159G 1.51 N24Y-G54A 1.11 R14L-R159S 1.52 N24Y-R127L 1.37 R14L-T39P1.50 N24Y-R127S 1.14 R14M-N24L-R159S- 1.15 N24Y-R127V 1.22 R123VN24Y-R159F 1.29 R14M-R159F 1.18 R127A-R159F 1.12 R14M-R159W 1.36R127H-R159Q 1.78 R14Q-R123F 1.58 R127H-R159T- 1.66 R14S-N24E-R127W 1.31S185F R14S-N24V 1.11 R14S-N24Y 1.12 N24L-S76T-A93G- 1.24 R14T-N24T-R127Q1.38 R127K-R159K R14T-R127Y 1.98 N24M-A93S-R127K- 1.23 R14V-N24D-R127C1.28 R159K R14V-N24G-P189S 1.14 N24M-A93T-R127K- 1.18 R14V-N24L-R127F1.14 R159K R14V-R127A 1.55 N24M-G54E-A93H- 1.35 R14V-R159F 1.43R127K-R159K R14V-R159W 1.24 N24M-G54E-S76N- 1.36 R14W-N24A 1.10A93S-R127K-R159K R14W-R123L 1.19 N24M-S76V-A93H- 1.16 R14W-R123V 1.40R127K-R159K R14W-R159V 1.15 N24Q-A93G-R127K- 1.10 R159V-G49D 1.67 R159KR159V-R123G 1.20 N24Q-G54D-S76L- 1.11 N024E-G049A- 1.29 A93G-R127K-A093H-R127K- R159K A143N-R159K- N24Q-G54I-S76T- 1.10 I181Q A93G-R127K-N024E-G049A- 1.11 R159K A093S-S099D- N24Q-G54Q-A93G- 1.10 R127K-A143N-R127K-R159K R159K-I181Q N24Q-S76A-A93G- 1.12 N024Q-G049A- 1.12R127K-R159K A093S-S099A- N24T-G54Q-S76N- 1.19 R127K-A143N- A93G-R127K-R159K-I181T R159K N24A-G54E-S76D- 1.41 N24T-G54Q-S76V- 1.20 A93G-R127K-R127K-R159K R159K N24T-S76I-R127K- 1.14 N24E-A93G-R127K- 1.32 R159KR159K N24W-A93G- 1.20 N24E-G54L-S76E- 1.15 R127K-R159K A93G-R127K-N24W-G54D-A93H- 1.21 R159K R127K-R159K N24E-G54Q-A93S- 1.46 R014I-S076A-1.21 R127K-R159K A093G-R127K- N24E-S76D-A93T- 1.35 R159K-I181TR127K-R159K R014I-S076A- 1.16 N24H-G54E-A93G- 1.10 A093H-R127K-R127K-R159K R159K-I181Q R014I-S076D- 1.10 R014M-S076A- 1.35 A093H-R127K-A093S-R127K- R159K-I181Q R159K-I181K R014I-S076D- 1.27 R014M-S076A- 1.15A093H-R127K- A093S-R127K- R159K-I181T R159K-I181T R014I-S076D- 1.31R014M-S076A- 1.28 A093S-R127K- A093T-R127K- R159K-I181T R159K-I181QR014I-S076E- 1.46 R014M-S076D- 1.31 A093S-R127K- A093S-R127K-R159K-I181Q R159K-I181T R014I-S076E- 1.20 R014M-S076E- 1.12 A093T-R127K-A093H-R127K- R159K-I181K R159K-I181T R014I-S076N- 1.13 R014M-S076E- 1.16A093H-R127K- A093S-R127K- R159K-I181Q R159K-I181T R014L-S076A- 1.21R014M-S076N- 1.12 A093H-R127K- A093G-R127K- R159K R159K-I181KR014L-S076A- 1.15 R014M-S076N- 1.19 A093H-R127K- A093G-R127K-R159K-I181Q R159K-I181T R014L-S076D- 1.16 R014M-S076N- 1.15 A093H-R127K-A093H-R127K- R159K-I181T R159K-I181T R014M-S076A- 1.30 R014M-S076N- 1.32A093G-R127K- A093S-R127K- R159K-I181T R159K-I181T

In additional experiments using liquid detergent, the following resultswere obtained. The variants indicated in the following table exhibitedactivity greater than wild-type plus one standard deviation (>1.1Performance Index).

TABLE 11-2 Wash Performance at Low pH BMI BMI Low pH Low pH DetergentDetergent [Perf. Variant [Perf. Ind.] Variant Ind.] T36S-R127Q-R159E1.24 G12D-S15E 1.71 S15E-R35E 1.15 G12D-R35H-R159E 1.74 S15E-R35D 1.20G12D-R35H-R123Q 1.89 S15E-R159E 1.24 G12D-R35H 2.38 S15E-R127Q 1.39G12D-R35E 1.85 S15E-R123Q 1.22 G12D-R35D 1.68 S15E-R123E 1.38 G12D-R159E1.32 R79T R127Q R179Q 1.23 G12D-R14Q-R35H 1.44 R35H-R159E 1.37G12D-R14I-R35H 1.54 R35F R61S R159Q 1.39 G65Q-R127A-R159K 1.10 R35FR159Q 1.49 R127A-R159K 1.35 R35D-R127Q 1.56 R14I-G65Q 1.38 R16Q R79TR159Q 1.42 R14I-G65Q-N67L- 1.14 R179Q R159K R16Q R79T R123L 1.62R14I-G65Q-R127A- 1.43 R16Q R79T 1.59 R14I-G65Q-R127A- 1.27 R16Q R35F1.29 R159K R16Q R159Q R179Q 1.31 R14I-G65Q-R159K 1.38 R16Q R159Q 1.26R14I-R127A 1.33 R14Q-T121E 1.37 R14I-R127A-R159K 1.44 R14Q-R35E 1.36R14I-R159K 1.32 R14Q-R35D 1.27 R14I-R35F 1.13 R14Q-R123Q 1.33R14I-R35F-G65Q 1.16 R14L R79T 1.59 R14I-R35F-R159K 1.28 R127Q-R159E 1.34R14I-S76V 1.40 R127Q R159Q 1.45 R35F-R127A-R159K 1.17 R123Q-R159E 1.51R123L R159Q 1.58 R123F-R159E 1.77

As indicated by these results, numerous multiply-substituted variantsperformed better than the wild-type ASP in this assay system.

Example 12 Cleaning Performanceo of ASP Variants in AutomaticDishwashing

In this Example, experiments conducted to determine the cleaningperformance of ASP variants in Automatic Dish Washing (ADW) TABLETACTION PACK® detergent in comparison with a benchmark serine protease(“Protease B”) on protease-sensitive stains described. As shown in theTable below, ASP variants removes stains much better than protease B.

A micro-washing test with 24 well plates was performed. Stainless steeldisks were punched out using a hammer and punch. The disks were cleanedand weighed, in order to obtain initial weights. Egg stains wereprepared as described below (“Scrambled Egg Preparation” and “Egg YolkPreparation”). Then, 50 uL of prepared egg were dispensed onto eachdisk. The preparations were allowed to dry at room temperature for 30minutes, then were baked in an oven at 80° C. for 2 hours. The diskswere cooled to room temperature. After soiling, the disks were weighedin order to obtain the soiled weights of the disks. The soiled diskswere inserted into NUNCLON® 24 well polystyrene plate. Auto dish washingproduct solution was prepared by thoroughly mixing the appropriateamount of ADW without enzyme product (e.g., Powder 4500 ppm) into 1 L of11 gpg water at 40° C. Hardness solution was prepared by mixing 188.57 gCaCl₂.2H₂O and 86.92 g MgCl₂.6H₂O into 1 L DI water. In the test, 2 mLof pre-warmed ADW solution were added at 55° C. to each well. Theappropriate amount of enzyme was then to each well. In some embodiments,the tests were run using six across, with four duplicates, while inother embodiments, the test were run four across with six duplicates.The plates were then sealed with film. The plates were placed in apre-warmed incubator/shaker and secured with flask clamps. Plates werewashed for 30 minutes at an appropriate temperature (e.g., 55° C. forEuropean wash conditions), at 180 RPM. After washing, the plates werecarefully rinsed by dipping them in a warm water bath three times. Thedisks were removed from the plates and dried in an oven for 1 hour at80° C. Once dry, the disks were weighed, in order to obtain the washeddisk weights. The gravimetric evaluation resulting in approximatepercent removal was calculated as follows:% Removal=(soiled weight−washed weight)/(soiled weight−clean weight)×100Removal Index=% Removal/% Removal by Benchmark Protease (Protease B)Scrambled Egg Preparation

Eggs for use in the testing of the protease and/or detergentcompositions comprising test enzyme(s) were prepared as follows. First,100 mls of 10% fat milk were mixed with 3 whole eggs. This mixture wascooked with continuous stirring until the mixture was slightly runny.Then, an additional 40 ml milk were added and the mixture was blendedwith a hand mixture or blender on high until smooth, with no signs oflumps. The egg mixture was allowed to cool to room temperature beforesoiling.

Egg Yolk Preparation

A water bath was heated by setting the temperature probe on the hotplate to 60° C. A pulp strainer was placed over a beaker. To prepare theegg yolks, 6-9 yolks were cracked and the yolks separated from thewhites. The yolk sacs were not broken, although any residue present onthe yolks was removed. The yolks were gently rinsed in cold water. Theyolks were gently broken over the strainer and beaker. Once all of theyolks have been strained, the beaker containing the strained yolks wasplaced in the water bath. The yolks were stirred for 30 minutes, at 60°C. The yolks were removed from the water bath and placed in a cool waterbath for 30 minutes. Any skin that formed on the surface was gentlyremoved.

Preparation of Egg Stains Using Stainless Steel Slides (1×3 Inch)

Each metal slide was dipped into the scrambled egg or egg yolkpreparation (prepared as described above) and tapped once, ensuring thatthere was approximately the same amount of egg on each slide. The backsof the slides were wiped on a tissue to clean them. The slides were thenplaced on two oven racks in numerical order. The slides were left to dryfor 30 minutes at room temperature. The slides were then cooked for 2hours at 80° C. (+/−1° C.), with the upper and lower shelves beingexchanged and rotated by 180° (+/−1° C.) after 1 hour. The cooled,soiled slides were weighed in order 1-96.

For washing, eight stained slides were placed both in top rack andbottom rack of the washing machine. Pre-wash and main wash was based onWhirlpool 840 cup capacity of 40 mls for pre-wash and 60 mls for mainwash. Typically in one test, four products were run using four GE 500machines. For the test cycle, soiled slides were loaded into themachines and test product placed in the pre-wash and main wash cups. Thewater temperature was set to 120° F. and the machines turned on thenormal wash cycle. The slides were removed from the machines forweighting at the end of the final rinse cycle. The slides did not gothrough the dry cycle. By the end of the fourth repetition, each productwas run once in each machine. All four repetitions were run in one day.The next day, this same procedure was repeated using new soiledsubstrates, to provide a total of 8 repetitions per product, which werethen averaged for the final results. The gravimetric evaluationresulting in approximated percent removal was calculated as follows:Removal=(soiled weight−washed weight)/(soiled weight−clean weight)×100Removal Index=% Removal/% Removal by Benchmark Protease (Protease B)

The results of these performance tests are provided below in Table 12-1:

TABLE 12-1 Stain Removal Index vs. Protease B in 24 Well Screening OnScrambled On Egg Enzyme Egg Yolk WT ASP 101 100 R14N-R127K-R159L 134 120R14I-A64K-T86K-N112E-R123F-D184T 126 124 G12D-R35E-G63R-R79K-T109M 124113 R14L 128 123 G12D-R35E 120 138 R14M-S76D-A93H-R127K-R159K-I181K 123103

Example 13 Cleaning Performance of ASP Variants in Powder LaundryDetergent

In this Example, experiments conducted to determine the cleaningperformance of ASP variants in laundry powder detergent ARIEL® aredescribed. In these experiments, the performance of the variants wascompared with a benchmark serine protease (e.g., “Protease C”) on RealItem Cleaning (RIC).

The tests are conducted using Meile washing machines using the followingconditions: temperature 40° C., hardness 21 gpg, detergent 7300 ppm,protease 0.88 ppm, R10.75 kg, total ballast and RI 2.5 kg, water 13 L,wash time of 20 minutes, rinse time of 3×5 minutes.

In these experiments, king and queen size sheets, and clean socks (J&RCoordinating Service) are quartered for clean ballast. In addition,Consumer Real Items (RI) are tested, including socks, t-shirts,pillowcases, towels, and tea towels. Soils comprising yeast and AS1(Artificial Soil 1; Equest) are used in these experiments. Thecomposition of AS1 is provided below in Table 13-1.

TABLE 13-1 Composition of AS1 Component % Artificial Sebum 14.5 Tea (PGTips) 6.5 Coffee (Nescafe) 4.0 Orange Juice 12.0 Tomato Sauce (Heinz)13.0 Grass 4.5 Chocolate (Heinz Baby) 7.5 Burnt Butter 2.5 Cooking Oil14.0 ETC Clay 5.0 NTC Clay 5.0 Hoover Dust 4.0 Make up 7.5

The garments are cut so as to ensure equal garment size for testing,with garments selected for uniform soil level. The two halves are codedin paneling pair order (e.g., left/right of garment).

Garment 1 Garment 2 Garment 3 Garment 4 Garment 5 Garment 6 Garment 7Garment 8 A B B A B A A B B A A B A B B A

Garments labeled “A” are washed with ASP variant, while Garments labeled“B” are washed with Protease C. The tests were conducted 4×4×2 (eachtreatment is repeated in four machines with two internal replicates ofRI).

Test Procedure

Ballast of clean quartered bed sheets and RI are combined to make atotal load weight of 2.5 kg and readied to be loaded into a manual Meilewashing machine, with the “21 gpg” spout on the front panel turned on.The washer is then turned on. The temperature is set to 40° C. and forthe short wash cycle. Next, the ballast and RI are added to the washingmachine. In addition, 20 g AS1 and clean socks stained with 17 g Bakersyeast are added to each replicate. Then, detergent (95 g) is added todetergent tray in the machine and the cycle started. Once the detergentis flushed from the tray, any liquids to be added are added. The machinethen runs through its cycles. When the washing cycle is completed, thecontents are transferred to a dryer and dried for 30 minutes. The itemsare then visually graded to provide PSU results and preference numbersin the LSG program.

Test Grading

The whole tests are graded in a single session within 72 hrs of washingand drying the test items. The split item garments are laid out in thepanel room and graded by at least 3 judges using the Scheffe scale.Additionally, the stains A vs B are graded by at least 2 judges.

Results Handling

Grades are entered into a hand-held Psion set up with the wash testsoftware. When grading of all split items has been completed, the dataare transferred into a personal computer where the average PSU grade, %preference between A and B treatments, and LSD for each garment arecalculated. Significant differences between products are calculated to aconfidence limit of 90%.

The PSU grading systems are used to compare two products (formulae). Thetwo formulae are tested on performance (e.g., post wash stainresiduals). In these experiments several fabrics, washed with bothproducts (e.g., product A and product B), are compared. Two or morejudges perform the grading, in which the following Schelle scale isused:

0: No preference

1: I think this product is a little better (unsure)

2: I know this product is a little better

3: This product is better

4: This product is much better

Example 14 Storage Stability of ASP Variants

In this Example, experiments conducted to determine the storagestability of various ASP variants tested in liquid TIDE® detergent incomparison with WT ASP are described. The protease activity of thevariants and WT ASP were tested using a Hitachi 911 Automatic Analyzer.In these tests succinyl-L-Ala-L-Pro-L-Phe-p-nitroanilide's (pNA) wasused to as the substrate to assess protease activity. In this test, theterminal carboxylamide bond is cleaved by active protease yieldingp-nitroaniline. The intensity of the resultant yellow color, read at 415nm/450 nm, is proportional to the activity of the protease in the samplewhich is calibrated with SAVINASE® protease standard.

As indicated in the following Table, there were significant improvementsin storage stability of the engineered variants as compared to WT ASP.In this Table, the “Retained Activity %” was determined using thefollowing formula: Retained Activity %=Activity/Initial Activity×100%

TABLE 14-1 Retained Activity in 28 Days Storage in Liquid TIDE ® at 90°F. 4 7 14 21 28 Initial 1 Day Days Days Days Days Days WT ASP 100% 91%32% 0 0 0 0 R18 100% 99% 41% 0 0 0 0 G12D-R35H 100% 100% 100% 100%  97%92% 87% G12D-R35E 100% 100% 100% 100%  100%  97% 94% R35E 100% 100% 100%96% 87% 79% 72% R35E-R18 100% 100% 100% 94% 82% 70% 61% R35D 100% 100%100% 86% 71% 56% 45%

Example 15 Determination of ASP Cleaning Activity

In this Example, experiments conducted to determine the cleaningactivity of ASP under various conditions, as well as the properties ofthe various wash conditions are described.

There is a wide variety of wash conditions including varying detergentformulations, wash water volume, wash water temperature, and length ofwash time. Thus, detergent components such as proteases must be able totolerate and function under adverse environmental conditions. Forexample, detergent formulations used in different areas have differentconcentrations of their relevant components present in the wash water.For example, a European detergent typically has about 3000-8000 ppm ofdetergent components in the wash water, while a Japanese detergenttypically has less than 800 (e.g., 667 ppm) of detergent components inthe wash water. In North America, particularly the United States,detergent typically have about 800 to 2000 (e.g., 975 ppm) of detergentcomponents present in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations, as they range from1500 ppm to 6000 ppm of detergent components in the wash water.Brazilian detergents typically has approximately 1500 ppm of detergentcomponents present in the wash water. However, other high suds phosphatebuilder detergent geographies, not limited to other Latin Americancountries, may have high detergent concentration systems up to about6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 800 ppm of detergent composition (“lowdetergent concentration geographies”), for example about 667 ppm inJapan, to between about 800 ppm to about 2000 ppm (“medium detergentconcentration geographies”), for example about 975 ppm in U.S. and about1500 ppm in Brazil, to greater than about 2000 ppm (“high detergentconcentration geographies”), for example about 3000 ppm to about 8000ppm in Europe and about 6000 ppm in high suds phosphate buildergeographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 975 ppm of detergent within the wash solution,about 62.79 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

As a further example, different geographies use different washtemperatures. The temperature of the wash water in Japan is typicallyless than that used in Europe. For example, the temperature of the washwater in North America and Japan can be between 10 and 30° C. (e.g.,about 20° C.), whereas the temperature of wash water in Europe istypically between 30 and 50° C. (e.g., about 40° C.).

As a further example, different geographies may have different waterhardness. Water hardness is typically described as grains per gallonmixed Ca²⁺/Mg²⁺. Hardness is a measure of the amount of calcium (Ca²⁺)and magnesium (Mg²⁺) in the water. Most water in the United States ishard, but the degree of hardness varies from area to area. Moderatelyhard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts permillion (i.e., parts per million converted to grains per U.S. gallon isppm # divided by 17.1 equals grains per gallon) of hardness minerals.Table 15-1 provides ranges of water hardness.

TABLE 15-1 Water Hardness Ranges Water Grains per Gallon Parts perMillion Soft less than 1.0 less than 17 Slightly hard 1.0 to 3.5 17 to60 Moderately hard 3.5 to 7.0  60 to 120 Hard  7.0 to 10.5 120 to 180Very hard greater than 10.5 greater than 180

European water hardness is typically greater than 10.5 (e.g., 10.5-20.0)grains per gallon mixed Ca²⁺/Mg²⁺ (e.g., about 15 grains per gallonmixed Ca²⁺/Mg²⁺). North American water hardness is typically greaterthan Japanese water hardness, but less than European water hardness. Forexample, North American water hardness can be between 3 to 10 grains,3-8 grains or about 6 grains. Japanese water hardness is typically lowerthan North American water hardness, typically less than 4, for example 3grains per gallon mixed Ca²⁺/Mg²⁺.

The present invention provides protease variants that provide improvedwash performance in at least one set of wash conditions and typically inmultiple wash conditions.

As described herein, the protease variants are tested for performance indifferent types of detergent and wash conditions using a microswatchassay (See above, and U.S. patent application Ser. No. 09/554,992; andWO 99/34011, both of which are incorporated by reference herein).Protease variants are tested for other soil substrates also in a similarfashion.

Example 16 Liquid Fabric Cleaning Compositions

This Example provides liquid fabric cleaning compositions that find usein conjunction with the present invention. These compositions arecontemplated to find particular utility under Japanese machine washconditions, as well as for applications involving cleaning of fineand/or delicate fabrics. Table 13-1 provides a suitable composition.However, it is not intended that the present invention be limited tothis specific formulation, as many other formulations find use with thepresent invention.

TABLE 16-1 Liquid Fabric Cleaning Composition Component Amount (%)AE2.5S 2.16 AS 3.30 N-Cocoyl N-methyl 1.10 glucamine Nonionic surfactant10.00 Citric acid 0.40 Fatty acid 0.70 Base 0.85 Monoethanolamine 1.011,2-Propanediol 1.92 EtOH 0.24 HXS 2.09 Protease.sup.1 0.01 Amylase 0.06Minors/inerts to 100%

TABLE 16-2 Liquid Laundry Detergent Compositions Detergent CompositionsComponent I II III IV V LAS 18.0 — 6.0 — — C₁₂-C₁₅AE_(1.8)S — 2.0 8.011.0 5.0 C₈-C₁₀ propyl dimethyl 2.0 2.0 2.0 2.0 1.0 amine C₁₂-C₁₄ alkyldimethyl — — — — 2.0 amine oxide C₁₂-C₁₅AS — 17.0 — 7.0 8.0 CFAA — 5.04.0 4.0 3.0 C₁₂-C₁₄ Fatty alcohol 12.0 6.0 1.0 1.0 1.0 ethoxylateC₁₂-C₁₈ Fatty acid 11.0 11.0 4.0 4.0 3.0 Citric acid (anhydrous) 5.0 1.03.0 3.0 2.0 DETPMP 1.0 1.0 1.0 1.0 0.5 Monoethanolamine 11.0 8.0 5.0 5.02.0 Sodium hydroxide 1.0 1.0 2.5 1.0 1.5 Percarbonate — 3.5 — 2.5 —Propanediol 12.7 14.5 13.1 10. 8.0 Ethanol 1.8 1.8 4.7 5.4 1.0 PectinLyase — — — 0.005 — Amylase — 0.002 — — — Cellulase — — 0.0002 — 0.0001Lipase 0.1 — 0.1 — 0.1 Protease A 0.05 0.3 0.055 0.5 0.2 DETBCHD — —0.02 0.01 — SRP1 0.5 0.5 — 0.3 0.3 Boric acid 2.4 2.4 2.8 2.8 2.4 Sodiumxylene sulfonate — — 3.0 — — DC 3225C 1.0 1.0 1.0 1.0 1.02-butyl-octanol 0.03 0.04 0.04 0.03 0.03 DTPA 0.5 0.4 0.35 0.28 0.4Brightener 1 0.18 0.10 0.11 — — ASP Variant 0.05 0.3 0.08 0.5 0.2Balance to 100% perfume/dye and/or water

TABLE 16-3 Liquid Laundry Detergent Compositions Detergent CompositionComponent I I II III IV V LAS 11.5 11.5 9.0 — 4.0 — C₁₂-C₁₅AE_(2.85)S —— 3.0 18.0 — 16.0 C₁₄-C₁₅E_(2.5)S 11.5 11.5 3.0 — 16.0 — C₁₂-C₁₃E₉ — —3.0 2.0 2.0 1.0 C₁₂-C₁₃E₇ 3.2 3.2 — — — — CFAA — — — 5.0 — 3.0 TPKFA 2.02.0 — 2.0 0.5 2.0 Citric Acid 3.2 3.2 0.5 1.2 2.0 1.2 (Anhydrous) Caformate 0.1 0.1 0.06 0.1 — — Na formate 0.5 0.5 0.06 0.1 0.05 0.05 NaCulmene 4.0 4.0 1.0 3.0 1.2 — Sulfonate Borate 0.6 0.6 — 3.0 2.0 3.0 Nahydroxide 6.0 6.0 2.0 3.5 4.0 3.0 Ethanol 2.0 2.0 1.0 4.0 4.0 3.0 1,2Propanediol 3.0 3.0 2.0 8.0 8.0 5.0 Mono- 3.0 3.0 1.5 1.0 2.5 1.0ethanolamine TEPAE 2.0 2.0 — 1.0 1.0 1.0 PB1 — 4.5 — 2.8 — Protease A0.03 0.03 0.01 0.03 0.02 0.02 Lipase — — — 0.002 — — Amylase — — — —0.002 — Cellulase — — — — — 0.0001 Pectin Lyase 0.005 0.005 — — — ASPVariant 0.03 0.05 0.01 0.03 0.08 0.02 SRP 1 0.2 0.2 — 0.1 — — DTPA — — —0.3 — — PVNO — — — 0.3 — 0.2 Brightener 1 0.2 0.2 0.07 0.1 — — Siliconeantifoam 0.04 0.04 0.02 0.1 0.1 0.1 Balance to 100% perfume/dye, and/orwater

TABLE 16-4 Liquid Laundry Detergent Compositions Composition Component III III IV V LAS 24.0 32.0 6.0 8.0 6.0 C₁₂-C₁₅AE_(1.8)S — — 8.0 11.0 5.0C₈-C₁₀ propyl dimethyl 2.0 2.0 2.0 2.0 1.0 amine C₁₂-C₁₄ alkyl dimethyl— — — — 2.0 amine oxide C₁₂-C₁₅AS — — 17.0 7.0 8.0 CFAA — 5.0 4.0 4.03.0 C₁₂-C₁₄ Fatty alcohol 12.0 6.0 1.0 1.0 1.0 ethoxylate C₁₂-C₁₈ Fattyacid 3.0 — 4.0 4.0 3.0 Citric acid (anhydrous) 6.0 5.0 3.0 3.0 2.0DETPMP — — 1.0 1.0 0.5 Monoethanolamine 5.0 5.0 5.0 5.0 2.0 Sodiumhydroxide — — 2.5 1.0 1.5 1N HCl aqueous solution #1 #1 — — —Propanediol 12.7 14.5 13.1 10. 8.0 Ethanol 1.8 2.4 4.7 5.4 1.0 DTPA 0.50.4 0.3 0.4 0.5 Pectin Lyase — — — 0.005 — Amylase 0.001 0.002 — — —Cellulase — — 0.0002 — 0.0001 Lipase 0.1 — 0.1 — 0.1 ASP Variant 0.050.3 0.08 0.5 0.2 Protease A — — — — 0.1 SRP1 0.5 0.5 — 0.3 0.3 Boricacid 2.4 2.4 2.8 2.8 2.4 Sodium xylene sulfonate — — 3.0 — — DC 3225C1.0 1.0 1.0 1.0 1.0 2-butyl-octanol 0.03 0.04 0.04 0.03 0.03 Brightener1 0.12 0.10 0.18 0.08 0.10 Balance to 100% perfume/dye and/or waterThe pH of Examples 16-4. compositions (I)-(II) is about 5 to about 7,and of 1(III)-(V) is about 7.5 to about 8.5. #1: add 1N HCl aq. soln toadjust the neat pH of the formula in the range from about 3 to about 5.

Example 17 Liquid Dishwashing Compositions

This Example provides liquid dishwashing compositions that find use inconjunction with the present invention. These compositions arecontemplated to find particular utility under Japanese dish washingconditions. However, it is not intended that the present invention belimited to this specific formulation, as many other formulations finduse with the present invention.

The following compact high density dishwashing detergent compositionsare provided by the present invention.

TABLE 17-1 Compact High Density Dishwashing Detergent CompositionsCompositions Component I II III IV V VI STPP — 45.0 45.0 — — 40.0 3NaCitrate 17.0 — — 50.0 40.2 — 2H₂O Na Carbonate 17.5 14.0 20.0 — 8.0 33.6Bicarbonate — — — 26.0 — — Silicate 15.0 15.0 8.0 — 25.0 3.6Metasilicate 2.5 4.5 4.5 — — — PB1 — — 4.5 — — — PB4 — — — 5.0 — —Percarbonate — — — — — 4.8 PAAC 0.02 0.05 0.03 0.04 0.03 0.05 BB1 — 0.10.1 — 0.5 — BB2 0.2 0.05 — 0.1 — 0.6 Nonionic 2.0 1.5 1.5 3.0 1.9 5.9HEDP 1.0 — — — — — DETPMP 0.6 — — — — — Paraffin 0.5 0.4 0.4 0.6 — —Protease B 0.072 0.053 0.053 0.026 0.059 0.01 Amylase 0.012 — 0.012 —0.021 0.006 Lipase — 0.001 — 0.005 — — Pectin Lyase 0.001 0.001 0.001 —— — ASP Variant 0.072 0.053 0.053 0.026 0.059 0.01 BTA 0.3 0.2 0.2 0.30.3 0.3 Poly- 6.0 — — — 4.0 0.9 carboxylate Perfume 0.2 0.1 0.1 0.2 0.20.2 Balance to 100% Moisture and/or Minors* *Brightener/Dye/SRP1/NaCarboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/HighMolecular PEG/Clay.The pH of Examples 17-1 compositions (I) through (VI) is from about 9.6to about 11.3.

The following hand dish liquid detergent compositions are provided bythe present invention.

TABLE 17-2 Hand Dish Liquid Detergent Compositions DetergentCompositions Component I II III IV V VI C₁₂-C₁₅AE_(1.8)S 30.0 28.0 25.0— 15.0 10.0 LAS — — — 5.0 15.0 12.0 Paraffin Sulfonate — — — 20.0 — —C₁₀-C₁₈ Alkyl 5.0 3.0 7.0 — — — Dimethyl Amine Oxide Betaine 3.0 — 1.03.0 1.0 — C₁₂ poly-OH fatty — — — 3.0 — 1.0 acid amide C₁₄ poly-OH fatty— 1.5 — — — — acid amide C₁₁E₉ 2.0 — 4.0 — — 20.0 DTPA — — — — 0.2 —Tri-sodium Citrate 0.25 — — 0.7 — — dihydrate Diamine 1.0 5.0 7.0 1.05.0 7.0 MgCl₂ 0.25 — — 1.0 — — Protease A 0.02 0.01 0.02 0.01 0.02 0.05Amylase 0.001 — — 0.002 — 0.001 Sodium Cumene — — — 2.0 1.5 3.0Sulphonate DETBCHD — — — 0.01 0.02 0.01 PB1 1.5 2.8 1.2 — — — ASPVariant 0.02 0.01 0.03 0.01 0.02 0.05 Balance to 100% perfume/dye and/orwater

The following liquid automatic dishwashing detergents compositions areprovided by the present invention.

TABLE 17-3 Liquid Automatic Dishwashing Detergent CompositionsCompositions Component I II III IV V STPP 16 16 18 16 16 PotassiumSulfate — 10 8 — 10 1,2 propanediol 6.0 0.5 2.0 6.0 0.5 Boric Acid 4.03.0 3.0 4.0 3.0 CaCl₂ dihydrate 0.04 0.04 0.04 0.04 0.04 Nonionic 0.50.5 0.5 0.5 0.5 Protease B 0.03 0.03 0.03 0.03 0.03 Amylase 0.02 — 0.020.02 — ASP Variant 0.1 0.03 0.05 0.03 0.06 Balance to 100% perfume/dyeand/or water

The following tablet detergent compositions are provided by the presentinvention. These compositions are prepared by compression of a granulardishwashing detergent composition at a pressure of 13KN/cm², using astandard 12 head rotary press.

TABLE 17-4 Tablet Detergent Compositions Compositions Component I II IIIIV V VI VII VIII STPP — 48.8 44.7 38.2 — 42.4 46.1 36.0 3Na Citrate 2H₂O20.0 — — — 35.9 — — — Na Carbonate 20.0 5.0 14.0 15.4 8.0 23.0 20.0 28.0Silicate 15.0 14.8 15.0 12.6 23.4 2.9 4.3 4.2 Lipase 0.001 — 0.01 — 0.02— — — Protease B 0.042 0.072 0.042 0.031 — — — — Protease C — — — —0.052 0.023 0.023 0.029 ASP Variant 0.01 0.08 0.05 0.04 0.052 0.0230.023 0.029 Amylase 0.012 0.012 0.012 — 0.015 — 0.017 0.002 Pectin Lyase0.005 — — 0.002 — — — — PB1 — — 3.8 — 7.8 — — 8.5 Percarbonate 6.0 — —6.0 — 5.0 — — PAAC 0.02 0.03 0.05 0.04 0.03 0.02 0.04 0.05 BB1 0.2 — 0.5— 0.3 0.2 — — BB2 — 0.2 — 0.5 — — 0.1 0.2 Nonionic 1.5 2.0 2.0 2.2 1.04.2 4.0 6.5 TAED — — — — — 2.1 — 1.6 HEDP 1.0 — — 0.9 — 0.4 0.2 — DETPMP0.7 — — — — — — — Paraffin 0.4 0.5 0.5 0.5 — — 0.5 — BTA 0.2 0.3 0.3 0.30.3 0.3 0.3 — Polycarboxylate 4.0 — — — 4.9 0.6 0.8 — PEG 400-30,000 — —— — — 2.0 — 2.0 Glycerol — — — — — 0.4 — 0.5 Perfume — — — 0.05 0.2 0.20.2 0.2 Balance to 100% Moisture and/or Minors* *Brightener/Dye/SRP1/NaCarboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/HighMolecular PEG/Clay.The pH of Examples 17-4.(I) through (VIII) is from about 10 to about11.5.The tablet weight of Examples 17-4.(I) through 7(VIII) is from about 20grams to about 30 grams.

The following dishwashing composition finds particular use underJapanese washing conditions.

TABLE 17-5 Liquid Dishwashing Compositions Component A B AE1.4S 24.6924.69 N-cocoyl N-methyl glucamine 3.09 3.09 Amine oxide 2.06 2.06Betaine 2.06 2.06 Nonionic surfactant 4.11 4.11 Hydrotrope 4.47 4.47Magnesium 0.49 0.49 Ethanol 7.2 7.2 LemonEase 0.45 0.45 Geraniol/BHT —0.60/0.02 Amylase 0.03 0.005 Protease 0.01 0.43 Balance to 100%

Example 18 Liquid Fabric Cleaning Compositions

The proteases of the present invention find particular use in cleaningcompositions. For example, it is contemplated that liquid fabriccleaning composition of particular utility under Japanese machine washconditions be prepared in accordance with the invention. In somepreferred embodiments, these compositions comprise the followingcomponents shown in Table 18-1.

TABLE 18-1 Liquid Fabric Cleaning Composition Component Amount (%)AE2.5S 15.00 AS 5.50 N-cocoyl N-methyl 5.50 glucamine Nonionicsurfactant 4.50 Citric acid 3.00 Fatty acid 5.00 Base 0.97Monoethanolamine 5.10 1,2-Propanediol 7.44 EtOH 5.50 HXS 1.90 Boric acid3.50 Ethoxylated 3.00 tetraethylenepentaimine SRP 0.30 Protease 0.069Amylase 0.06 Cellulase 0.08 Lipase 0.18 Brightener 0.10 Minors/inerts to100%

Example 19 Granular Fabric Cleaning Compositions

In this Example, various granular fabric cleaning compositions that finduse with the present invention are provided. The following Tablesprovide suitable compositions. However, it is not intended that thepresent invention be limited to these specific formulations, as manyother formulations find use with the present invention.

TABLE 19-1 Granular Fabric Cleaning Compositions Formulations ComponentA B C D Protease1 0.10 0.20 0.03 0.05 Protease2 0.2 0.15 C13 linearalkyl benzene sulfonate 22.00 22.00 22.00 22.00 Phosphate (as sodiumtripolyphosphate) 23.00 23.00 23.00 23.00 Sodium carbonate 23.00 23.0023.00 23.00 Sodium silicate 14.00 14.00 14.00 14.00 Zeolite 8.20 8.208.20 8.20 Chelan (diethylaenetriamine-petaacetic acid) 0.40 0.40 0.400.40 Sodium sulfate 5.50 5.50 5.50 5.50 Water Balance to 100%

TABLE 19-2 Granular Fabric Cleaning Compositions Formulations ComponentA B C D Protease1 0.10 0.20 0.30 0.05 Protease2 0.2 0.1 C12 alkylbenzene sulfonate 12.00 12.00 12.00 12.00 Zeolite A (1-10 micrometer)26.00 26.00 26.00 26.00 C12-C14 secondary (2,3) alkyl sulfate, 5.00 5.005.00 5.00 Na salt Sodium citrate 5.00 5.00 5.00 5.00 Optical brightener0.10 0.10 0.10 0.10 Sodium sulfate 17.00 17.00 17.00 17.00 Fillers,water, minors Balance to 100%

The following laundry compositions are provided by the presentinvention. These compositions are suitable for use as granules ortablets.

TABLE 19-3 Granule and Tablet Laundry Detergent Compositions CompositionComponent I II III IV V Base Product C₁₄-C₁₅AS or TAS 8.0 5.0 3.0 3.03.0 LAS 8.0 — 8.0 — 7.0 C₁₂-C₁₅AE₃S 0.5 2.0 1.0 — — C₁₂-C₁₅E₅ or E₃ 2.0— 5.0 2.0 2.0 QAS — — — 1.0 1.0 Zeolite A 20.0 18.0 11.0 — 10.0 SKS-6(dry add) — — 9.0 — — MA/AA 2.0 2.0 2.0 — — AA — — — — 4.0 3Na Citrate2H₂O — 2.0 — — — Citric Acid (Anhydrous) 2.0 — 1.5 2.0 — DTPA 0.2 0.2 —— — EDDS — — 0.5 0.1 — HEDP — — 0.2 0.1 — PB1 3.0 4.8 — — 4.0Percarbonate — — 3.8 5.2 — NOBS 1.9 — — — — NACA OBS — — 2.0 — — TAED0.5 2.0 2.0 5.0 1.00 BB1 0.06 — 0.34 — 0.14 BB2 — 0.14 — 0.20 —Anhydrous Na Carbonate 15.0 18.0 8.0 15.0 15.0 Sulfate 5.0 12.0 2.0 17.03.0 Silicate — 1.0 — — 8.0 Protease B 0.033 0.033 — — — Protease C — —0.033 0.046 0.033 Lipase — 0.008 — — — Amylase 0.001 — — — 0.001Cellulase — 0.0014 — — — Pectin Lyase 0.001 0.001 0.001 0.001 0.001 ASPVariant 0.03 0.05 1.0 0.06 0.1 Balance to 100% Moisture and/or Minors**Perfume/Dye, Brightener/SRP1/NaCarboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/HighMolecular PEG/Clay.

The following laundry detergent compositions are contemplated to provideparticular use under European machine wash conditions.

TABLE 19-3 Granular Fabric Cleaning Compositions Formulations ComponentA B C LAS 7.0 5.61 4.76 TAS 1.57 C45AS 6.0 2.24 3.89 C25E25 1.0 0.761.18 C45E7 2.0 C25E3 4.0 5.5 QAS 0.8 2.0 2.0 STPP Zeolite 25.0 19.5 19.5Citric acid 2.0 2.0 2.0 NaSKS-6 8.0 10.6 10.6 Carbonate I 8.0 10.0 8.6MA/AA 1.0 2.6 1.6 CMC 0.5 0.4 0.4 PB4 12.7 Percarbonate 19.7 TAED 3.15.0 Citrate 7.0 DTPMP 0.25 0.2 0.3 HEDP 0.3 0.3 0.3 QEA 1 0.9 1.2 1.0Protease 1 0.02 0.05 0.035 Lipase 0.15 0.25 0.15 Cellulase 0.28 0.280.28 Amylase 0.4 0.7 0.3 PVPI/PVNO 0.4 0.1 Photoactivated 15 ppm 27 ppm27 ppm bleach (ppm) Brightener 1 0.08 0.19 0.19 Brightener 2 0.04 0.04Perfume 0.3 0.3 0.3 Effervescent 15 15 5 granules (malic acid 40%,sodium bicarbonate 40%, sodium carbonate 20%) Silicon 0.5 2.4 2.4antifoam Minors/inerts to Balance to 100% 100%

Example 20 Hard Surface Cleaning Detergent Compositions

The present invention also provides compositions suitable for cleaningof hard surfaces.

TABLE 20 Liquid Hard Surface Cleaning Detergent Compositions CompositionComponent I II III IV V VI VII C₉-C₁₁E₅ 2.4 1.9 2.5 2.5 2.5 2.4 2.5C₁₂-C₁₄E₅ 3.6 2.9 2.5 2.5 2.5 3.6 2.5 C₇-C₉E₆ — — — — 8.0 — — C₁₂-C₁₄E₂₁1.0 0.8 4.0 2.0 2.0 1.0 2.0 LAS — — — 0.8 0.8 — 0.8 Sodium culmene 1.52.6 — 1.5 1.5 1.5 1.5 sulfonate Isachem ® AS 0.6 0.6 — — — 0.6 — Na₂CO₃0.6 0.13 0.6 0.1 0.2 0.6 0.2 3Na Citrate 2H₂O 0.5 0.56 0.5 0.6 0.75 0.50.75 NaOH 0.3 0.33 0.3 0.3 0.5 0.3 0.5 Fatty Acid 0.6 0.13 0.6 0.1 0.40.6 0.4 2-butyl octanol 0.3 0.3 — 0.3 0.3 0.3 0.3 PEG DME-2000 ® 0.4 —0.3 0.35 0.5 — — PVP 0.3 0.4 0.6 0.3 0.5 — — MME PEG (2000) ® — — — — —0.5 0.5 Jeffamine ® ED-2001 — 0.4 — — 0.5 — — PAAC — — — 0.03 0.03 0.03— DETBCHD 0.03 0.05 0.05 — — — — Protease B 0.07 0.05 0.05 0.03 0.060.01 0.04 Amylase 0.12 0.01 0.01 — 0.02 — 0.01 Lipase — 0.001 — 0.005 —0.005 — ASP Variant 0.07 0.05 0.08 0.03 0.06 0.01 0.04 MCAEM 3.5 5.6 4.85.3 3.6 8.0 4.7 (C₈-C₁₀E₂ Acetate) Pectin Lyase 0.001 — 0.001 — — —0.002 PB1 3.5 4.6 2.7 3.8 3.6 4.2 2.7 Balance to 100% perfume/dye,and/or water

The pH of these compositions is from about 7.4 to about 9.5.

Example 21 Animal Feed Comprising ASP

The present invention also provides animal feed compositions comprisingASP variants. In this Example, one such feed, suitable for poultry isprovided. However, it is not intended that the present invention belimited to this specific formulation, as the proteases of the presentinvention find use with numerous other feed formulations. It is furtherintended that the feeds of the present invention be suitable foradministration to any animal, including but not limited to livestock(e.g., cattle, pigs, sheep, etc.), as well as companion animals (e.g.,dogs, cats, horses, rodents, etc.). The following Table provides aformulation for a mash, namely a maize-based starter feed suitable foradministration to turkey poults up to 3 weeks of age.

TABLE 21-1 Animal Feed Composition Ingredient Amount (wt. %) Maize 36.65Soybean meal (45.6% CP) 55.4 Animal-vegetable fat 3.2 Dicalciumphosphate 2.3 Limestone 1.5 Mineral premix 0.3 Vitamin premix 0.3 Sodiumchloride 0.15 DL methionine 0.2

In some embodiments, this feed formulation is supplemented with variousconcentrations of the protease(s) of the present invention (e.g., 2,000units/kg, 4,000 units/kg and 6,000 units/kg).

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

Those of skill in the art readily appreciate that the present inventionis well adapted to carry out the objects and obtain the ends andadvantages mentioned, as well as those inherent therein. Thecompositions and methods described herein are representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It is readily apparent to oneskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

What is claimed is:
 1. An isolated serine protease variant of theCellulomonas 69B4 protease set forth in SEQ ID NO: 8 wherein the variantconsists of at least 90% amino acid identity to SEQ ID NO: 8, comprisessubstitutions at R127 and R159, and has the serine protease substrateactivity of the Cellulomonas 69B4 protease set forth by SEQ ID NO:
 8. 2.A composition comprising the isolated serine protease variant ofclaim
 1. 3. A composition comprising the isolated serine proteasevariant of claim 1, wherein said isolated serine protease variant hasimmunological cross-reactivity with an antibody to the serineCellulomonas 69B4 protease set forth by the amino acid sequence of SEQID NO:
 8. 4. The variant of claim 1, wherein said variant has improvedthermostability as compared to the Cellulomonas 69B4 protease set forthby the amino acid sequence of SEQ ID NO:
 8. 5. The variant of claim 1,wherein said variant has improved linear alkylbenzene sulfonate (LAS)stability as compared to the Cellulomonas 69B4 protease set forth by theamino acid sequence of SEQ ID NO:
 8. 6. The variant of claim 1 whereinsaid variant has enhanced protease activity as measured by PerformanceIndex (PI), as compared to the Cellulomonas 69B4 protease set forth bythe amino acid sequence of SEQ ID NO:
 8. 7. The variant of claim 6,wherein said variant has improved caseinolytic activity as compared tothe Cellulomonas 69B4 protease set forth by the amino acid sequence ofSEQ ID NO:
 8. 8. The variant of claim 6, wherein said variant hasimproved keratinolytic activity as compared to the Cellulomonas 69B4protease set forth by the amino acid sequence of SEQ ID NO:
 8. 9. Thevariant of claim 1, wherein said variant has improved wash performanceactivity as compared to the Cellulomonas 69B4 protease set forth by theamino acid sequence of SEQ ID NO:
 8. 10. The variant of claim 9, whereinsaid variant has improved dishwashing performance activity as comparedto the Cellulomonas 69B4 protease set forth by the amino acid sequenceof SEQ ID NO:
 8. 11. The variant of claim 9, wherein said variant hasimproved stain removal activity as compared to the Cellulomonas 69B4protease set forth by the amino acid sequence of SEQ ID NO:
 8. 12. Acleaning composition comprising at least one serine protease variant asset forth in claim
 1. 13. A cleaning composition comprising at least onevariant serine protease of claim 1, wherein said serine protease varianthas immunological cross-reactivity with an antibody to the serineCellulomonas 69B4 protease set forth by the amino acid sequence of SEQID NO:
 8. 14. The cleaning composition of claim 12, further comprisingone or more additional enzymes selected from the group consisting ofproteases, amylases, lipases, mannanases, pectinases, cutinases,oxidoreductases, hemicellulases, and cellulases.
 15. The cleaningcomposition of claim 12, further comprising a stabilizing agent selectedfrom the group consisting of borax, glycerol, and competitiveinhibitors.
 16. The cleaning composition of claim 12, wherein saidserine protease variant is an autolytically stable variant.
 17. Acleaning composition comprising at least 0.0001 weight percent of theserine protease variant of claim
 1. 18. The cleaning composition ofclaim 17, wherein said composition comprises from about 0.001 to about0.5 weight percent of said serine protease variant.
 19. The cleaningcomposition of claim 17, wherein said composition comprises from about0.01 to about 0.1 weight percent of said serine protease variant. 20.The cleaning composition of claim 12, wherein said cleaning compositionis selected from liquid, powder, granular, and tablet compositions. 21.The cleaning composition of claim 12, wherein said composition furthercomprises a hydrogen peroxide source.
 22. The cleaning composition ofclaim 21, wherein said hydrogen peroxide source comprises at least onepersalt, wherein said persalt is alkalimetal perborate, alkalimetalpercarbonate, alkalimetal perphosphate, alkalimetal persulfate, or amixture thereof.
 23. The cleaning composition of claim 22, wherein saidcomposition further comprises a bleach catalyst, bleach activator and/ormixtures thereof.